Scalable process for protein purification

ABSTRACT

The invention provides a process for the purification recombinantly expressed, self-assembled VLP from the homogenate of a bacterial host, wherein the process can be scaled up to a commercial production scale in a cost effective manner. The process comprises a first chromatography using an anion exchange matrix, a second chromatography using hydroxyapatite and, optionally, a size exclusion chromatography. VLP preparations obtained by the process of the invention are essentially free of endotoxin contaminations.

FIELD OF THE INVENTION

This invention is related to the field protein purification. Provided isa process for the preparative purification of recombinantly expressed,self-assembled virus-like-particles (VLPs) from bacterial homogenates,wherein the process can be scaled up to a commercial production scaleand wherein the process allows for efficient removal of endotoxincontaminations and other host cell derived impurities from the VLPpreparation.

BACKGROUND OF THE INVENTION

Recent vaccination strategies exploit the immunogenicity of viruses orvirus-like-particles (VLPs) to enhance the immune response towardsantigens. For example, WO02/056905 demonstrates the utility of VLPs ascarriers to present antigens linked thereto in highly ordered repetitiveantigen arrays. Such antigen arrays can cause a strong immune response,in particular antibody responses, against the antigen, including selfantigens. Furthermore, VLPs have been shown to be useful in the therapyof diseases as delivery means for immunostimulatory substances(WO2003/024481). VLPs are therefore useful in the production ofpharmaceuticals for the treatment of infectious diseases and allergiesas well as for the efficient induction of self-specific immuneresponses, e.g. for the treatment of cancer, rheumatoid arthritis andvarious other diseases. For the production of VLP based pharmaceuticalsefficient processes for expression and purification of VLPs arerequired.

For reasons of scalability and efficiency and, thus, cost saving, VLPcarriers for the manufacture of pharmaceuticals are preferably producedby recombinant gene expression in a prokaryotic expression system. Viralcapsid proteins have been shown to efficiently self-assemble to formVLPs upon expression in a bacterial host. For example, hepatitis B virusderived VLP has been obtained by expression of HBc protein in E. coliand purification of the VLP from bacterial homogenate on a sucrosegradient (WO01/85208). VLPs of bacteriophages, preferably of RNAbacteriophages, are particularly suited as antigen carriers and havebeen produced in E. coli, wherein the assembled VLPs were isolated fromcrude bacterial homogenates by various methods.

For example, recombinant VLP derived from bacteriophage fr was isolatedfrom lysed E. coli cells by ammonium sulphate precipitation followed bysize exclusion chromatography using a SEPHADEX® G100 column with aSEPHADEX® G25 pre-column (Pushko et al. 1993, Protein Engineering6(8)883-891). Soluble recombinant VLPs derived from bacteriophage MS-2were isolated from lysed E. coli cells by a combination of ammoniumsulphate precipitation and separation on a sucrose density gradient,while less soluble variants were isolated by size exclusionchromatography (Mastico et al. 1993, Journal of General Virology74:541-548). WO92/13081 teaches the isolation of MS-2 derived VLP byfractionated ammonium sulphate precipitation combined with eithersucrose density gradient separation, gel filtration or immuno affinitychromatography. A multi step purification scheme for recombinant MS-2derived VLP comprising ammonium sulphate precipitation, isoelectricpoint precipitation, sucrose density gradient separation and sizeexclusion chromatography was also reported (Stockley & Mastico 2000,Methods in Enzymology 326:551-569). Recombinant VLPs derived frombacteriophage Qβ have been purified from bacterial homogenate by sizeexclusion chromatography using a SEPHAROSE® column (Kozlovska et al.1993, Gene 137:133-137) or by a combination of fractionated ammoniumsulphate precipitation and size exclusion chromatography with SEPHAROSE®CL-4B (Vasiljeva et al 1998, FEBS Letters 431:7-11; Ciliens et al. 2000,FEBS Letters 24171:1-4).

Proteins isolated from bacterial homogenates are typically contaminatedwith endotoxins and other host cell derived impurities, such as hostcell DNA and host cell proteins. The presence of host cell derivedimpurities, especially endotoxins, is generally undesired inpharmaceutical preparations. Endotoxins are lipopolysaccharides whichare invariably associated with the outer membrane of gram-negativebacteria, such as E. coli. They show a strong toxic, inflammatory and/orimmunogenic effect on mammals, including humans, when entering the bloodstream. Thus, removal of even minute amounts of endotoxins from proteinpreparations used for the manufacture of a pharmaceutical composition isessential. The processes which so far have been applied for thepurification of recombinant VLPs from bacterial homogenates are notcapable of reliably removing endotoxin contaminants to an extent whichis acceptable for pharmaceutical compositions and/or said processescomprise steps, such as sucrose gradient separation, which can hardly bescaled up for commercial production of the VLP.

It is an object of the present invention to provide a process for thepurification of recombinantly expressed, self-assembled VLPs frombacterial homogenate, wherein the VLPs are essentially free of host cellderived impurities, especially of endotoxins, and wherein the processcan be scaled up to a commercial production scale in a cost effectivemanner.

The assembly of the VLP takes place in the cytosol of the bacterial hostexpressing the VLP, while endotoxins are associated with the cell wallof the bacterial host. Therefore, endotoxins are typically notencapsulated inside the VLP and can thus be efficiently removed by theprocess of the invention. The VLP preparations obtained by the processof the invention typically comprise endotoxin contaminations atconcentrations which are about 50 times lower than those observed inpreparations obtained by the methods mentioned above.

The invention therefore provides a process for the purification of a VLPfrom a recombinant bacterial host expressing said VLP, wherein theprocess is capable of removing endotoxins and that fraction of nucleicacids and host cell proteins which is not encapsulated inside the VLP.

SUMMARY OF THE INVENTION

One embodiment of the invention is a process for the purification of aVLP from a recombinant bacterial host expressing said VLP, the processcomprising a first chromatography using a first chromatography matrix,preferably a hydroxyapatite matrix or an anion exchange matrix, a secondchromatography using a second chromatography matrix, preferably ahydroxyapatite matrix, and, optionally, a final purification step, alsoreferred to as “polishing step”, comprising at least one thirdchromatography, wherein preferably said at least one thirdchromatography is size exclusion chromatography. It has surprisinglybeen found that the combination of said first and said secondchromatography provides for high purity VLP preparations, in particularfor very efficient removal of endotoxins, wherein scalability of theprocess is maintained. It has further been found, that the removal ofsaid endotoxins is most efficient, when said second chromatographymatrix is a hydroxyapatite matrix.

One embodiment of the invention is a process for the purification of aVLP from a recombinant bacterial host expressing said VLP, the processcomprising the steps of: (a) homogenizing said bacterial host; (b)clarifying the homogenate obtained by said homogenizing; (c) purifyingsaid VLP from the clarified homogenate obtained by said clarifying in afirst chromatography comprising the steps of: (i) binding said VLP to afirst chromatography matrix; (ii) washing said first chromatographymatrix; and (iii) eluting said VLP from said first chromatographymatrix; and (d) further purifying said VLP from the eluate obtained bysaid first chromatography in a second chromatography, wherein saidsecond chromatography is performed on a second chromatography matrix,wherein said second chromatography matrix is a hydroxyapatite matrix;wherein said steps are performed in the given order.

In a further embodiment said second chromatography comprises the stepsof: (i) binding said VLP to said second chromatography matrix, whereinsaid second chromatography matrix is a hydroxyapatite matrix; (ii)washing said second chromatography matrix; and (iii) eluting said VLPfrom said second chromatography matrix; wherein said steps are performedin the given order.

In a further embodiment said process additionally comprising the step offinally purifying said VLP obtained by said second chromatography by atleast one third chromatography, wherein said at least one thirdchromatography is selected from: (a) hydrophobic interactionchromatography (HIC); (b) immobilized metal ions affinity chromatography(IMAC); and (c) size exclusion chromatography.

In a further embodiment said VLP comprises capsid protein of a virusselected from the group consisting of: (a) RNA bacteriophage; (b)bacteriophage; (c) Hepatitis B virus; (d) measles virus; (e) Sindbisvirus; (f) rotavirus; (g) foot-and-mouth-disease virus; (h) Norwalkvirus; (i) Alpha Virus; (j) retrovirus; (k) retrotransposon Ty; (l)human Papilloma virus; (m) Polyoma virus; (n) Tobacco mosaic virus; and(o) Flock House Virus.

In a further embodiment said clarifying of said homogenate is performedby a method selected from the group consisting of: (a) centrifugation;(c) tangential flow filtration, preferably using a filter having amembrane comprising a pore size of about 0.45 μm; and (c) a combinationof (a) and (c).

In a further embodiment said first chromatography matrix is an anionexchange matrix, preferably an anion exchange matrix comprising TMAEgroups.

In a further embodiment said first chromatography matrix is a tentacleanion exchange matrix comprising (i) resin particles of cross-linkedmethacrylate polymer or cross-linked vinyl polymer (ii) acrylamidetentacles, wherein said acrylamide tentacles are attached to the surfaceof said resin particles, and wherein said acrylamide tentacles aresubstituted with TMAE (Trimethylaminoethyl-) groups.

In a further embodiment said first chromatography matrix is selectedfrom the group consisting of: (a) FRACTOGEL® EMD TMAE (M), preferablyhaving a particle size of 40-90 μm; (b) FRACTOGEL® EMD TMAE Hicap (M),preferably having a particle size of 40-90 μm; (c) FRACTOPREP® DEAE,preferably having a particle size of 30-150 μm; (d) MACRO-PREP® CHTCeramic Hydroxyapatite Type I, preferably having a particle size ofabout 80 μm; (e) MACRO-PREP® CHT Ceramic Hydroxyapatite Type II,preferably having a particle size of about 80 μm; (f) MATREX® GranularSilica PEI-300 Å, preferably having a particle size of 35-70 μm; (g)MATREX® Granular Silica PEI-1000 Å, preferably having a particle size of35-70 μm; (h) POROS® 50 HQ; (i) CIM-QA (quarternary amino group, BIASeparations Cat. No. 210.5113), and (j) CIM-DEAE.

In a further embodiment said first chromatography comprises the stepsof: (i) equilibrating said first chromatography matrix with a firstequilibration buffer; (ii) binding said VLP to a first chromatographymatrix; (iii) washing said first chromatography matrix with a firstwashing buffer; and (iv) eluting said VLP from said first chromatographymatrix with a first elution puffer; wherein said first equilibrationbuffer, said first washing buffer and said first elution buffer comprisean inorganic salt, preferably an alkaline metal halogenide, morepreferably potassium chloride or sodium chloride, most preferably sodiumchloride.

In a further embodiment said first equilibration buffer comprises atmost about 200 mM sodium chloride, said first washing buffer comprisesabout 425 mM sodium chloride, and said first elution buffer comprisesleast about 500 mM sodium chloride or a gradient of sodium chloride,wherein preferably said gradient is from at most about 400 to at leastabout 650 mM sodium chloride, preferably from 425 to 650 mM sodiumchloride.

In a further embodiment said first equilibration buffer, said firstwashing buffer and said first elution buffer comprise a pH of about 7.2,wherein preferably said pH is stabilized by a phosphate buffer, morepreferably by about 20 mM phosphate buffer, most preferably by about 20mM sodium phosphate buffer.

In a further embodiment said hydroxyapatite matrix is a ceramichydroxyapatite matrix, wherein preferably said ceramic hydroxyapatitematrix comprises a particle size of about 80 μm and a pore size of theparticles of about 800-1000 Å, wherein further preferably said ceramichydroxyapatite matrix is MACRO-PREP® CHT Ceramic Hydroxyapatite Type II.

In a further embodiment said second chromatography comprises the stepsof: (i) equilibrating said second chromatography matrix with a secondequilibration buffer; (ii) binding said VLP to said secondchromatography matrix, wherein said second chromatography matrix is ahydroxyapatite matrix; (ii) washing said second chromatography matrixwith a second washing buffer; and (iii) eluting said VLP from saidsecond chromatography matrix; wherein said second equilibration buffer,said second washing buffer and said second elution buffer, comprise aninorganic salt, preferably an alkaline metal halogenide, more preferablypotassium chloride or sodium chloride, most preferably sodium chloride.

In a further embodiment said second equilibration buffer comprises about100 to 400 mM sodium chloride, said second washing buffer comprisesabout 150 mM sodium chloride, and said second elution buffer comprises900 mM sodium chloride and about 200 mM sodium phosphate buffer.

In a further embodiment said second equilibration buffer, said secondwashing buffer and said second elution buffer comprise a pH of about7.2, wherein preferably said pH is stabilized by a phosphate buffer,preferably by a sodium phosphate buffer.

In a further embodiment said clarifying further comprises the step ofexposing said VLP to oxidative conditions.

In a further embodiment said least one third chromatography is at leastone, preferably exactly one, size exclusion chromatography, wherein saidsize exclusion chromatography is preferably performed using a gelfiltration matrix selected from the group consisting of: (a) SEPHADEX®G-25; (b) SEPHAROSE® CL-4B; and (c) SEPHACRYL®-S400.

In a further embodiment the invention provides a process for thepurification of a VLP of RNA bacteriophage Qβ from a recombinantbacterial host expressing said VLP, the process comprising the steps of:(a) homogenizing said bacterial host; (b) clarifying the homogenateobtained by said homogenizing, wherein said clarifying further comprisesthe step of exposing said VLP to oxidative conditions; (c) purifyingsaid VLP from the clarified homogenate obtained by said clarifying in afirst chromatography comprising the steps of: (i) equilibrating atentacle anion exchange matrix, wherein said equilibrating is performedwith a first equilibration buffer, wherein said first equilibrationbuffer comprises about 150 mM sodium chloride and a pH of 7.2; (ii)binding said VLP to said tentacle anion exchange matrix; (iii) washingsaid tentacle anion exchange matrix, wherein said washing is performedwith a first washing buffer comprising about 425 mM sodium chloride anda pH of 7.2; and (iv) eluting said VLP from said tentacle anion exchangematrix, wherein said eluting is performed with a first elution buffercomprising a gradient of 425 to 650 mM sodium chloride and a pH of 7.2;wherein preferably said tentacle anion exchange matrix is a tentacleanion exchange matrix as defined above, most preferably FRACTOGEL® EMDTMAE (M); (d) further purifying said VLP from the eluate obtained bysaid first chromatography in a second chromatography comprising thesteps of: (i) equilibrating a hydroxyapatite matrix wherein saidequilibrating is performed with a second equilibration buffer comprisingabout 150 mM sodium chloride and a pH of 7.2; (ii) binding said VLP tohydroxyapatite matrix, preferably in the presence of about 250 mM sodiumchloride; (iii) washing said hydroxyapatite matrix, wherein said washingis performed with a second washing buffer comprising about 150 mM sodiumchloride and a pH of 7.2; (iv) eluting said VLP from said hydroxyapatitematrix, wherein said eluting is performed with a second elution buffercomprising about 900 mM sodium chloride, about 200 mM sodium phosphatebuffer and a pH of 7.2; wherein preferably said hydroxyapatite matrix isa hydroxyapatite matrix as defined above, most preferably a MACRO-PREP®CHT Ceramic Hydroxyapatite Type II matrix; (e) finally purifying saidVLP contained in the eluate of said second chromatography by exactly onesize exclusion chromatography, wherein said size exclusionchromatography is performed in the presence of about 150 mM sodiumchloride, and wherein further said size exclusion chromatography isperformed using a SEPHAROSE® CL-4B gel filtration matrix; wherein saidsteps are performed in the given order.

In a further embodiment the invention provides a process for thepurification of a VLP of RNA bacteriophage AP205 from a recombinantbacterial host expressing said VLP, the process comprising the steps of:(a) homogenizing said bacterial host; (b) clarifying the homogenateobtained by said homogenizing, wherein said clarifying further comprisesthe step of exposing said VLP to oxidative conditions; (c) purifyingsaid VLP from the clarified homogenate obtained by said clarifying in afirst chromatography comprising the steps of: (i) equilibrating atentacle anion exchange matrix, wherein said equilibrating is performedwith a first equilibration buffer, wherein said first equilibrationbuffer comprises about 150 mM sodium chloride and a pH of 7.2; (ii)binding said tentacle anion exchange matrix; (iii) washing said tentacleanion exchange matrix, wherein said washing is performed with a firstwashing buffer comprising about 425 mM sodium chloride and a pH of 7.2;and (iv) eluting said VLP from said tentacle anion exchange matrix,wherein said eluting is performed with a first elution buffer comprisingabout 550 mM sodium chloride and a pH of 7.2; wherein preferably saidtentacle anion exchange matrix is a tentacle anion exchange matrix asdefined above, most preferably FRACTOGEL® EMD TMAE (M); (d) furtherpurifying said VLP from the eluate obtained by said first chromatographyin a second chromatography comprising the steps of: (i) equilibrating ahydroxyapatite matrix, wherein said equilibrating is performed with asecond equilibration buffer comprising, about 100 mM sodium chloride andabout 5 mM sodium phosphate buffer and a pH of 7.2; (ii) binding saidVLP to said hydroxyapatite matrix, preferably in the presence of about100 mM sodium chloride and about 5 mM sodium phosphate buffer; (iii)washing said hydroxyapatite matrix wherein said washing is performedwith a second washing buffer comprising about 100 mM sodium chloride,about 5 mM sodium phosphate buffer and a pH of 7.2; (iv) eluting saidVLP from said hydroxyapatite matrix, wherein said eluting is performedwith a second elution buffer comprising about 250 mM sodium chloride,about 50 mM sodium phosphate buffer and a pH of 7.2; wherein preferablysaid hydroxyapatite matrix is a hydroxyapatite matrix as defined above,most preferably a MACRO-PREP® CHT Ceramic Hydroxyapatite Type II matrix;(e) finally purifying said VLP contained in the eluate of said secondchromatography by exactly one size exclusion chromatography, whereinsaid size exclusion chromatography is performed in the presence of about150 mM sodium chloride, and wherein further said size exclusionchromatography is performed using a SEPHAROSE® CL-4B gel filtrationmatrix; wherein said steps are performed in the given order.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

“one”, “a”, or “an”: When the terms “one”, “a”, or “an” are used in thisdisclosure, they mean “at least one” or “one or more”, unless otherwiseindicated.

“About”: The term “about” as used herein in connection with a numericalvalue refers to a range of ±10% of said value. E.g. a concentration ofabout 100 mM refers to a range of concentration of 100 mM±10%, i.e. 90to 110 mM; a concentration of at least about 100 mM refers to aconcentration which is not below 90 mM.

“Homogenate”: The term “homogenate” of a bacterial host relates to asuspension of bacteria cells, wherein at least 50%, preferably at least75%, more preferably at least 90%, still more preferably at least 95%,most preferably at least 99% of the bacteria cells have been disruptedby physical and/or enzymatic means. Disruption of the bacteria cellscan, for example, be achieved by sonication, by passage through a highpressure liquid homogenizer like the APV LAB 1000, by passage through aFrench press, by grinding with aluminium oxide and/or by lysozymetreatment.

“Coat protein”/“capsid protein”: The term “coat protein” and theinterchangeably used term “capsid protein” within this applicationrefers to a viral protein, preferably a subunit of a natural capsid of avirus, preferably of a RNA bacteriophage, which is capable of beingincorporated into a virus capsid or a VLP. For example, the specificgene product of the coat protein gene of RNA bacteriophage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” or “capsid proteins”of bacteriophage Qβ comprise the “Qβ CP” as well as the A1 protein.

“Fragment of a protein”: The term “fragment of a protein”, in particularfragment of a recombinant protein or recombinant capsid protein, as usedherein, is defined as a polypeptide, which is of at least 70%,preferably at least 80%, more preferably at least 90%, even morepreferably at least 95% the length of the wild-type recombinant protein,or capsid protein, respectively and which preferably retains thecapability of forming VLP. Preferably the fragment is obtained by atleast one internal deletion, at least one truncation or at least onecombination thereof. The term “fragment of a recombinant protein” or“fragment of a capsid protein” shall further encompass polypeptide,which has at least 80%, preferably 90%, even more preferably 95% aminoacid sequence identity with the “fragment of a recombinant protein” or“fragment of a coat protein”, respectively, as defined above and whichis preferably capable of assembling into a virus-like particle.

“Mutant recombinant protein”/“mutant of a recombinant protein”: Theterms “mutant recombinant protein” and “mutant of a recombinant protein”as interchangeably used in this application, or the terms “mutant coatprotein” and “mutant of a coat protein”, as interchangeably used in thisapplication, refer to a polypeptide having an amino acid sequencederived from the wild type recombinant protein, or coat protein,respectively, wherein the amino acid sequence is at least 80%,preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wildtype sequence and preferably retains the ability to assemble into a VLP.

“Polypeptide”: As used herein the term “polypeptide” refers to a polymercomposed of amino acid residues, generally natural amino acid residues,linked together through peptide bonds. Although a polypeptide may notnecessarily be limited in size, the term polypeptide is often used inconjunction with peptide of a size of about ten to about 50 amino acids.

“Protein”: As used herein, the term protein refers to a polypeptidegenerally of a size of above 20, more particularly of above 50 aminoacid residues. Proteins generally have a defined three dimensionalstructure although they do not necessarily need to, and are oftenreferred to as folded, in opposition to peptides and polypeptides whichoften do not possess a defined three-dimensional structure, but rathercan adopt a large number of different conformations, and are referred toas unfolded. The defined three-dimensional structures of proteins isespecially important for the association between the core particle andthe antigen, mediated by the second attachment site, and in particularby way of chemical cross-linking between the first and second attachmentsite using a chemical cross-linker. The amino acid linker is alsointimately related to the structural properties of proteins in someaspects of the invention.

“Recombinant coat protein”/“recombinant capsid protein”: A capsidprotein which is synthesised by a recombinant host cell.

“Recombinant bacterial host”: As used herein, the term “recombinantbacterial host” refers to a bacteria cell, preferably an E. coli cell,into which one or more nucleic acid molecules encoding have beenintroduced, wherein said nucleic acid molecule or nucleic acid moleculesencode a capsid protein forming the VLP to be purified by the process ofthe invention.

“Recombinant VLP”: The term “recombinant VLP”, as used herein, refers toa VLP that is obtained by a process which comprises at least one step ofrecombinant DNA technology. The term “VLP recombinantly produced”, asused herein, refers to a VLP that is obtained by a process whichcomprises at least one step of recombinant DNA technology. Thus, theterms “recombinant VLP” and “VLP recombinantly produced” areinterchangeably used herein and should have the identical meaning.

“RNA bacteriophage”: As used herein, the term “RNA bacteriophage” refersto an RNA virus infecting bacteria, preferably to single-strandedpositive-sense RNA viruses infecting bacteria.

“Sequence identity”: The amino acid sequence identity of polypeptidescan be determined conventionally using known computer programs such asthe Bestfit program. When using Bestfit or any other sequence alignmentprogram, preferably using Bestfit, to determine whether a particularsequence is, for instance, 95% identical to a reference amino acidsequence, the parameters are set such that the percentage of identity iscalculated over the full length of the reference amino acid sequence andthat gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed. This aforementionedmethod in determining the percentage of identity between polypeptides isapplicable to all proteins, polypeptides or a fragment thereof disclosedin this invention.

“Tentacle anion exchange matrix”: The expressions “tentacle anionexchange matrix” as used herein refers to an anion exchange matriximplementing the tentacle technology typically and preferably asdisclosed in WO96/22316, WO97/49754, EP0337144, DE4334359 or WO95/09695.Anion exchange matrices implementing the tentacle technology are resinparticles comprising, preferably on their surface, spacers formed bylinear polymer chains (tentacles), wherein said tentacles aresubstituted with functional groups having anion exchange activity.

Preferred tentacle anion exchange matrices are based on resins ofcopolymers on a methacrylate basis or on resins of vinyl polymers.Specifically preferred tentacle ion exchange matrices are FRACTOGEL® EMDTMAE ion exchangers and Fractoprep® DEAE ion exchangers (Merck), themost preferred tentacle anion exchange matrix is FRACTOGEL® ion EMDTMAE.

“Virus-like particle (VLP)”: as used herein, the term “virus-likeparticle” refers to a structure resembling a virus particle or it refersto a non-replicative or non-infectious, preferably a non-replicative andnon-infectious virus particle, or it refers to a non-replicative ornon-infectious, preferably a non-replicative and non-infectiousstructure resembling a virus particle, preferably a capsid of a virus.The term “non-replicative”, as used herein, refers to being incapable ofreplicating the genome comprised by the VLP. The term “non-infectious”,as used herein, refers to being incapable of entering the host cell.Preferably a virus-like particle in accordance with the invention isnon-replicative and/or non-infectious since it lacks all or part of theviral genome or genome function. Typically, a virus-like particle lacksall or part of the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome. A typical and preferredembodiment of a virus-like particle in accordance with the presentinvention is a viral capsid such as the viral capsid of thecorresponding virus, bacteriophage, preferably RNA bacteriophage. Theterms “viral capsid” or “capsid”, refer to a macromolecular assemblycomposed of viral protein subunits. Typically, there are 60, 120, 180,240, 300, 360 and more than 360 viral protein subunits. Typically andpreferably, the interactions of these subunits lead to the formation ofviral capsid or viral-capsid like structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA bacteriophages or HBcAgs have aspherical form of icosahedral symmetry.

“Virus-like particle of a RNA bacteriophage”: As used herein, the term“virus-like particle of a RNA bacteriophage” refers to a virus-likeparticle comprising, or preferably consisting essentially of orconsisting of coat proteins, mutants or fragments thereof, of a RNAbacteriophage. In addition, virus-like particle of a RNA bacteriophageresembling the structure of a RNA bacteriophage, being non replicativeand/or non-infectious, and lacking at least the gene or genes encodingfor the replication machinery of the RNA bacteriophage, and typicallyalso lacking the gene or genes encoding the protein or proteinsresponsible for viral attachment to or entry into the host. PreferredVLPs derived from RNA bacteriophages exhibit icosahedral symmetry andconsist of 180 subunits. Within this present disclosure the term“subunit” and “monomer” are interchangeably and equivalently used withinthis context. In this application, the term “RNA bacteriophage” and theterm “RNA-bacteriophage” are interchangeably used. A preferred method torender a virus-like particle of a RNA bacteriophage non replicativeand/or non-infectious is by genetic manipulation.

The invention relates to a process for the purification ofself-assembled virus-like particles (VLPs) from a homogenate of abacterial host, wherein said VLPs were produced by expression of one ormore viral capsid proteins in said bacterial host. VLPs derived from anyvirus known in the art may be purified by the process of the invention.Illustrative DNA or RNA viruses, the coat or capsid protein of which canbe used for the preparation of VLPs have been disclosed in WO2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page28, line 4 to page 31, line 4. Almost all commonly known viruses havebeen sequenced and the genes encoding their coat proteins are availableto the artisan. The preparation of VLPs by recombinantly expressingviral coat protein in a host is within the common knowledge of a skilledartisan.

One embodiment of the invention is a process for the purification of aVLP from a recombinant bacterial host expressing said VLP, the processcomprising the steps of: (a) homogenizing said bacterial host; (b)clarifying the homogenate obtained by said homogenizing; (c) purifyingsaid VLP from the clarified homogenate obtained by said clarifying in afirst chromatography comprising the steps of: (i) binding said VLP to afirst chromatography matrix; (ii) washing said first chromatographymatrix; and (iii) eluting said VLP from said first chromatographymatrix; and (d) further purifying said VLP from the eluate obtained bysaid first chromatography in a second chromatography, wherein saidsecond chromatography is performed on a second chromatography matrix,wherein said second chromatography matrix is a hydroxyapatite matrix;wherein said steps are performed in the given order.

In a preferred embodiment said second chromatography is a subtractivechromatography, wherein contaminants are bound to said secondchromatography matrix in the presence of an inorganic salt, and whereinthe concentration of said inorganic salt prevents the binding of saidVLP to said second chromatography matrix. In a further preferredembodiment said second chromatography is a subtractive chromatography,wherein preferably said second chromatography matrix is a hydroxyapatitematrix, and wherein further preferably said inorganic salt is analkaline metal halogenide, preferably potassium chloride or sodiumchloride, most preferably sodium chloride, and wherein furtherpreferably the concentration of said inorganic salt is at least about400 mM.

A further embodiment of the invention is a process for the purificationof a VLP from a recombinant bacterial host expressing said VLP, theprocess comprising the steps of: (a) homogenizing said bacterial host;(b) clarifying the homogenate obtained by said homogenizing; (c)purifying said VLP from the clarified homogenate obtained by saidclarifying in a first chromatography comprising the steps of: (i)binding said VLP to a first chromatography matrix; (ii) washing saidfirst chromatography matrix; and (iii) eluting said VLP from said firstchromatography matrix; and (d) further purifying said VLP from theeluate obtained by said first chromatography in a second chromatographycomprising the steps of: (i) binding said VLP to a second chromatographymatrix, wherein preferably said second chromatography matrix is ahydroxyapatite matrix; (ii) washing said second chromatography matrix;and (iii) eluting said VLP from said second chromatography matrix;wherein said steps are performed in the given order.

In one embodiment, said VLP comprises, or alternatively essentiallyconsists of, or consists of, recombinant proteins, mutants or fragmentsthereof, of a virus, wherein said virus preferably is a RNA virus, morepreferably a single stranded RNA virus, still more preferably a singlestranded positive sense RNA virus, most preferably a RNA bacteriophage.

In a further embodiment, said VLP comprises, or alternativelyessentially consists of, or consists of, recombinant proteins, mutantsor fragments thereof, of a virus selected form the group consisting of:(a) RNA bacteriophages; (b) bacteriophages; (c) Hepatitis B virus,preferably its capsid protein (Ulrich, et al., Virus Res. 50:141-182(1998)) or its surface protein (WO 92/11291); (d) measles virus (Warnes,et al., Gene 160:173-178 (1995)); (e) Sindbis virus; (f) rotavirus (U.S.Pat. No. 5,071,651 and U.S. Pat. No. 5,374,426); (g)foot-and-mouth-disease virus (Twomey, et al., Vaccine 13:1603 1610,(1995)); (h) Norwalk virus (Jiang, X., et al., Science 250:1580 1583(1990); Matsui, S. M., et al., J. Clin. Invest. 87:1456 1461 (1991));(i) Alphavirus; (j) retrovirus, preferably its GAG protein (WO96/30523); (k) retrotransposon Ty, preferably the protein p1; (l) humanPapilloma virus (WO 98/15631); (m) Polyoma virus; (n) Tobacco mosaicvirus; (o) Flock House Virus, (p) cowpea mosaic virus (CPMV), (q) cowpeachlorotic mottle virus (CCMV), and a virus of the genus Sobemovirus.

In one preferred embodiment, said VLP comprises, or alternativelyessentially consists of, or consists of more than one amino acidsequence, preferably two amino acid sequences, of the recombinantproteins, mutants or fragments thereof (referred to as mosaic VLP).

In a further preferred embodiment, said VLP is a VLP of Hepatitis Bvirus. The preparation of Hepatitis B virus-like particles has beendisclosed, inter alia, in WO00/32227, WO01/85208 and in WO02/056905. Ina preferred embodiment the VLP is composed of HBcAg (SEQ ID NO:1). Othervariants of HBcAg suitable for use in the practice of the presentinvention have been disclosed in page 34-39 of WO01/056905. In a furtherpreferred embodiment of the invention, a lysine residue is introducedinto the HBcAg polypeptide, to mediate the linking of an antigen to theVLP of HBcAg. In preferred embodiments, VLPs and compositions of theinvention are prepared using a HBcAg comprising, or alternativelyessentially consisting of, or consisting of amino acids 1-144, or 1-149,1-185 of SEQ ID NO:1, which is modified so that the amino acids atpositions 79 and 80 are replaced with a peptide having the amino acidsequence of Gly-Gly-Lys-Gly-Gly. This modification changes the SEQ IDNO:1 to SEQ ID NO:2. In further preferred embodiments, the cysteineresidues at positions 48 and 110 of SEQ ID NO:2, or its correspondingfragments, preferably 1-144 or 1-149, are mutated to serine. Theinvention further relates to the purification of VLPs comprising oralternatively consisting of Hepatitis B core protein mutants havingabove noted corresponding amino acid alterations. The invention furtherincludes VLPs, comprising HBcAg polypeptides which comprise, oralternatively consist of, amino acid sequences which are at least 80%,85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:2.

In one preferred embodiment of the invention, the virus-like particlecomprises, consists essentially of, or alternatively consists of,recombinant coat proteins, mutants or fragments thereof, of a RNAbacteriophage, wherein preferably, said RNA bacteriophage is selectedfrom the group consisting of (a) bacteriophage BZ13, (b) bacteriophageGA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophageTH1, (e) bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr,(h) bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17,(k) bacteriophage PP7, (l) bacteriophage FI, (m) bacteriophage ID2, (n)bacteriophage NL95, (o) bacteriophage SP, (p) bacteriophage TW28, (q)bacteriophage Qβ, (r) bacteriophage M11, (s) bacteriophage MX1, (t)bacteriophage ST, (u) bacteriophage TW18, and (v) bacteriophage VK.

In a further preferred embodiment said RNA bacteriophage is selectedfrom the group consisting of: (a) bacteriophage Qβ, (b) bacteriophageR17, (c) bacteriophage fr, (d) bacteriophage GA, (e) bacteriophage SP,(f) bacteriophage MS2, (g) bacteriophage M11, (h) bacteriophage MX1, (i)bacteriophage NL95, (k) bacteriophage f2, (l) bacteriophage PP7, and (m)bacteriophage AP205.

In a further preferred embodiment said RNA bacteriophage is selectedfrom the group consisting of: (a) bacteriophage Qβ; (b) bacteriophageR17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP;(f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i)bacteriophage NL95; (j) bacteriophage f2; (k) bacteriophage PP7 and (l)bacteriophage AP205.

In one preferred embodiment said VLP comprises coat protein, mutants orfragments thereof, of RNA bacteriophages, wherein preferably said coatprotein has an amino acid sequence selected from the group consistingof: (a) SEQ ID NO:3 □ referring to Qβ CP; (b) a mixture of SEQ ID NO:3and SEQ ID NO:4 (Qβ A1 protein); (c) SEQ ID NO:5 (R17 capsid protein);(d) SEQ ID NO:6 (fr capsid protein); (e) SEQ ID NO:7 (GA capsidprotein); (f) SEQ ID NO:8 (SP capsid protein); (g) a mixture of SEQ IDNO:8 and SEQ ID NO:9; (h) SEQ ID NO:10 (MS2 capsid protein); (i) SEQ IDNO:11 (M11 capsid protein); (j) SEQ ID NO:12 (MX1 capsid protein); (k)SEQ ID NO:13 (NL95 capsid protein); (l) SEQ ID NO:14 (f2 capsidprotein); (m) SEQ ID NO:15 (PP7 capsid protein); and (n) SEQ ID NO:16(AP205 capsid protein).

In one preferred embodiment of the invention, said VLP is a mosaic VLPcomprising or alternatively consisting of more than one amino acidsequence, preferably two amino acid sequences, of coat proteins, mutantsor fragments thereof, of a RNA bacteriophage. In one very preferredembodiment, said VLP comprises or alternatively consists of twodifferent coat proteins of a RNA bacteriophage, said two coat proteinshave an amino acid sequence of SEQ ID NO:3 and SEQ ID NO:4, or of SEQ IDNO:8 and SEQ ID NO:9. In preferred embodiments of the present invention,said VLP comprises, or alternatively consists essentially of, oralternatively consists of recombinant coat proteins, mutants orfragments thereof, of the RNA-bacteriophage Qβ, fr, AP205 or GA.

In one preferred embodiment, said VLP is a VLP of RNA bacteriophage Qβ.The capsid or virus-like particle of Qβ shows an icosahedral phage-likecapsid structure with a diameter of 25 nm and T=3 quasi symmetry. Thecapsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4:543-5554 (1996)), leading to a remarkable stabilityof the Qβ capsid. Capsids or VLPs made from recombinant Qβ coat proteinmay contain, however, subunits which are either not linked via disulfidebonds to other subunits within the capsid, or which are incompletelylinked, meaning that they comprise less than the maximum number ofpossible disulfide bonds.

Further preferred virus-like particles of RNA bacteriophages, inparticular of Qβ and fr in accordance of this invention are disclosed inWO 02/056905, the disclosure of which is herewith incorporated byreference in its entirety. Particular Example 18 of WO 02/056905 gavedetailed description of preparation of VLP particles from Qβ.

Upon expression in E. coli, the N-terminal methionine of Qβ coat proteinis usually removed (Stoll, E. et al., J. Biol. Chem. 252:990-993(1977)). VLP composed of Qβ coat proteins where the N-terminalmethionine has not been removed, or VLPs comprising a mixture of Qβ coatproteins where the N-terminal methionine is either cleaved or presentare also within the scope of the present invention.

In another preferred embodiment, said VLP is a VLP of RNA bacteriophageAP205. Assembly-competent mutant forms of AP205 VLPs, including AP205coat protein with the substitution of proline at amino acid 5 tothreonine, may also be used in the practice of the invention and leadsto other preferred embodiments of the invention. WO 2004/007538describes, in particular in Example 1 and Example 2, how to obtain VLPcomprising AP205 coat proteins, and hereby in particular the expressionand the purification thereto.

In one preferred embodiment, said VLP comprises or alternativelyessentially consists of, or consists of a mutant coat protein of avirus, preferably of a RNA bacteriophage, wherein said mutant coatprotein has been modified by removal of at least one lysine residue byway of substitution and/or by way of deletion. In another preferredembodiment, said VLP comprises or alternatively essentially consists of,or consists of a mutant coat protein of a virus, preferably of a RNAbacteriophage, wherein said mutant coat protein has been modified byaddition of at least one lysine residue by way of substitution and/or byway of insertion. The deletion, substitution or addition of at least onelysine residue allows varying the degree of coupling with an antigen.

VLPs or capsids of Qβ coat protein display a defined number of lysineresidues on their surface, with a defined topology with three lysineresidues pointing towards the interior of the capsid and interactingwith the RNA, and four other lysine residues exposed to the exterior ofthe capsid. Preferably, the at least one first attachment site is alysine residue, pointing to or being on the exterior of the VLP.

The purification of Qβ mutants, of which exposed lysine residues arereplaced by arginines is also encompassed by the present invention.Preferably, these mutant coat proteins comprise or alternativelyessentially consist of, or consist of an amino acid sequence selectedfrom the group of (a) Qβ-240 (SEQ ID NO:17, Lys13→Arg); (b) Qβ-243 (SEQID NO:18, Asn10→Lys); (c) Qβ-250 (SEQ ID NO:19, Lys2→Arg); (d) Qβ-251(SEQ ID NO:20, Lys16→Arg); and (e) Qβ-259 (SEQ ID NO:21, Lys2→Arg,Lys16→Arg). The construction, expression and purification of the aboveindicated Qβ mutant coat proteins, mutant Qβ coat protein VLPs andcapsids, respectively, are described in WO02/056905. In particular ishereby referred to Example 18 of above mentioned application.

In a further preferred embodiment said VLP comprises or alternativelyessentially consists of, or consists of a capsid protein ofbacteriophage AP205 having the amino acid sequence depicted in SEQ IDNO:16 or a mutation thereof, which is capable of forming a VLP,preferably the proteins AP205 P5T (SEQ ID NO:22) or AP205 N14D (SEQ IDNO:23).

In a very preferred embodiment said VLP comprises the 132 amino acidcoat protein C of E. coli RNA bacteriophage Qβ having the amino acidsequence depicted in SEQ ID NO:3 (133 amino acids with methionine inposition 1).

In a further preferred embodiment said VLP comprises a nucleic acidwhich is encapsulated inside said VLP, wherein preferably said nucleicacid is DNA or RNA, most preferably RNA, and wherein further preferablythe amount of said nucleic acid, preferably of said RNA, is at least 5μg, at least 10 μg, at least 20 μg, at least 30 μg at least 40 μg per100 μg of capsid protein. In a further preferred embodiment said amountof said nucleic acid, preferably of said RNA, is 5 to 60 μg, morepreferably 10 to 50 μg, still more preferably 20 to 40 μg, mostpreferably 25 to 35 μg per 100 μg of capsid protein.

In one embodiment, said bacterial host is an E. coli strain, preferablyan E. coli strain selected from the group consisting of RB791, DH20,Y1088, W3110 and MG1655. Most preferably, said bacterial host is E. coliRB791.

In one embodiment the process of the invention is performed at atemperature of 0 to 10° C., preferably of 2 to 8° C., most preferably ofabout 5° C.

The process of the invention comprises the steps of (a) homogenizingsaid bacterial host; (b) clarifying the homogenate obtained by saidhomogenizing; (c) purifying said VLP from the clarified homogenateobtained by said clarifying in a first chromatography, (d) furtherpurifying said VLP from the eluate obtained by said first chromatographyin a second chromatography, and, optionally, (e) further purifying saidVLP from the eluate of said second chromatography by at least one thirdchromatography, wherein preferably said at least one thirdchromatography is a size exclusion chromatography. In a preferredembodiment the steps of (a) homogenizing said bacterial host and (b)clarifying the homogenate obtained by said homogenizing are performed ata temperature of 0 to 10° C., preferably of 2 to 8° C., most preferablyof about 5° C. In a further preferred embodiment said purifying of saidVLP from the clarified homogenate, said further purifying of said VLPfrom the eluate obtained by said first chromatography, and/or saidfurther purifying of said VLP contained in the eluate of said secondchromatography are performed at room temperature, preferably at 15 to35° C., more preferably at 18 to 26° C., still more preferably at 20 to24° C., most preferably at 22° C.

The process of the invention comprises the step of homogenizing saidbacterial host. Cells of said bacterial host are harvested, e.g. bycentrifugation, and optionally stored at −80° C. Said homogenizing saidbacterial host is performed by disrupting the cells of said bacterialhost by physical, chemical or enzymatic means or by a combinationthereof. Preferably said homogenizing is performed by disrupting thecell wall of said bacterial host by sonication, by passage through ahigh pressure liquid homogenizer like, for example, APV LAB 1000, bypassage through a French press, or by grinding with aluminium oxide.Alternatively or additionally, preferably additionally, saidhomogenizing is performed by destabilizing the cell wall of saidbacterial host by detergent, preferably by sodium dodecyl sulphate(SDS), or, more preferably, by non-ionic detergents, preferably selectedfrom TRITON® X-100, TRITON® X-114, TWEEN® 20, IGEPAL® CA 630, BRIJ®35and mixtures thereof. In a very preferred embodiment said detergent isTRITON® X-100. Said detergent is preferably applied in a concentrationof 0.01 to 30%, more preferably 0.01 to 5%, most preferably about 0.1%.Alternatively or additionally, said homogenizing is performed bydestabilizing the cell wall of said bacterial host by exposure to a cellwall degrading enzyme, most preferably lysozyme.

The disruption of the bacteria cells is improved when the cellsuspension is passed through a high pressure liquid homogenizerrepeatedly. In a preferred embodiment said homogenizing said bacterialhost is performed by passing said bacterial host through a high pressureliquid homogenizer at least once, preferably at least twice, morepreferably at least three times, most preferably three times. The usageof a high pressure liquid homogenizer significantly improves thescalability of the process as it can be operated in a continuous mode.In a preferred embodiment, said homogenizing is performed by suspendingsaid bacterial host in a suspension buffer and passing the suspensionthrough a high pressure liquid homogenizer, preferably APV LAB 1000, ata pressure of 300 to 1200 bar, preferably 500 to 900 bar, morepreferably 600 to 800 bar and most preferably about 700 bar. The timerequired for homogenization in continuous mode needs to be adapted tothe volume and the cell density of the cell suspension. Criteria forsufficient homogenization of the bacterial host are the percentage ofcells remaining intact as observed, for example, by microscopy, or theconcentration of capsid protein detectable in the supernatant aftercentrifugation. Said suspension buffer preferably comprises an alkalinepH of about 8, an agent capable of forming complexes with metal ions,preferably EDTA, most preferably 1-50 mM EDTA, and a detergent,preferably selected from SDS, TWEEN®-20 or TRITON® X-100, mostpreferably TRITON® X-100, wherein the concentration of the detergent isabout 0.01 to 1.0%, more preferably about 0.05 to 0.5%, most preferablyabout 0.1%. In a very preferred embodiment said suspension buffercomprises a pH of 8.0, 5 mM EDTA and 0.1% (w/w) TRITON® X-100. In afurther preferred embodiment said suspension buffer comprises a cellwall degrading enzyme, most preferably lysozyme.

The process of the invention further comprises the step of clarifyingthe homogenate obtained by said homogenizing, wherein i.e. cell debrisis removed from the homogenate by either filtration or centrifugation.In one embodiment said homogenate is diluted before said clarifying isperformed. In a preferred embodiment said clarifying is performed byfiltering said homogenate in a tangential flow filtration (see Example2), preferably by tangential flow filtration using a filter suitable forthe processing of high viscosity media, preferably a filter cassettewith an open channel configuration, wherein further preferably saidfilter, preferably said filter cassette with an open channelconfiguration, is equipped with a membrane having a pore size of 0.2 to1.0 μm, preferably of 0.3 to 0.6 μm, more preferably of about 0.45 μm,most preferably 0.45 μm. In a further, equally preferred embodiment saidclarifying is performed by centrifuging said homogenate (see Example 3),wherein preferably said homogenate is exposed to an acceleration of atleast 7,000×g, more preferably at least 10,000×g for a period of timewhich is sufficient for the complete sedimentation of the cell debris.The required centrifugation time depends on the volume of the homogenateand the given technical set up. The artisan is able to determine therequired centrifugation time empirically which is required to obtain asufficiently solid pellet. Tangential flow filtration as well ascentrifugation allow for efficient scale-up of the process. To obtain aclarified homogenate which is essentially free of cell debris it isadvantageous to perform said clarifying of said homogenate by acombination of centrifugation and filtration. In a preferred embodimentsaid clarifying of said homogenate is achieved by a method selected fromthe group of (a) centrifuging said homogenate, (b) filtering saidhomogenate by tangential flow filtration, and (c) a combination of (a)and (b). In a very preferred embodiment said method is a combination ofcentrifuging said homogenate and filtering said homogenate by tangentialflow filtration, wherein preferably first said homogenate is clarifiedby centrifuging said homogenate and second the supernatant obtained bysaid centrifuging is further clarified by filtering said supernatant bytangential flow filtration (see Example 3a). Optionally, said clarifiedhomogenate is further clarified by sterile filtration to removeremaining cell debris and other particles. Therefore, in a furtherembodiment said clarifying further comprises the step of sterilefiltering the supernatant obtained by said centrifuging and/or thefiltrate obtained by said filtering by tangential flow filtration,preferably the filtrate obtained by said filtering by tangential flowfiltration, through a sterile filter having a pore size of about 0.18 to0.25 μm, preferably 0.20 to 0.22 μm, most preferably 0.22 μm.

The stability of many VLPs is established in a large extend by disulfidebounds between the capsid proteins forming said VLP. Generally,disulfide bounds are formed under oxidative and released under reductiveconditions. It has been found that the stability of a VLP, preferably ofa VLP comprising thiol groups capable of forming disulfide bounds suchas, for example, VLP of bacteriophage Qβ, can be significantly increasedby exposing said VLP to oxidative conditions prior to said purifyingsaid VLP from said clarified homogenate. This can, for example, beachieved by agitating said clarified homogenate containing said VLPunder condition allowing access of oxygen. In a further preferredembodiment said clarifying of said homogenate further comprises the stepof exposing said VLP to oxidative conditions, wherein said exposing ofsaid VLP to oxidative conditions is preferably performed after saidcentrifuging, after said filtering by tangential flow filtration orafter said sterile filtering, wherein preferably said VLP is a VLPcomprising thiol groups capable of forming disulfide bounds, morepreferably a VLP of a RNA bacteriophage, still more preferably VLP ofRNA bacteriophage AP205 or Qβ, most preferably VLP of RNA bacteriophageQβ. In a preferred embodiment said exposing of said VLP to oxidativeconditions is performed by slowly agitating said clarified homogenateunder conditions allowing the access of oxygen, wherein preferably saidagitating is performed at low temperature, preferably at 3 to 15° C.,more preferably at 4 to 10° C., most preferably at 8° C. The timerequired for the complete formation of disulfide bounds mainly dependson the efficiency of oxygen introduction into said clarified homogenateand can be determined by analysing samples of said VLP by SDS PAGE undernon-reducing conditions or by Ellman's test allowing the quantificationof free thiol groups. Volumes of 500 ml up to 10001 are preferablyagitated for 1 h up to 16 h, most preferably for 8 to 10 h. Theformation of disulfide bounds can further be improved by exposing saidVLP to slightly alkaline conditions. In a preferred embodiment saidexposing is performed in a buffer comprising a pH of 7.2 to 8.2, morepreferably of 7.2 to 7.8, still more preferably 7.3 to 7.7, still morepreferably 7.4 to 7.6, most preferably 7.5, wherein said pH refers to20° C. In a further preferred embodiment said exposing of said VLP tooxidative conditions is performed in a buffer comprising 20 to 100 mM,preferably 30 to 70 mM, more preferably 40 to 60 mM, most preferably 50mM Tris-HCl, and 1 to 25 mM, preferably 1 to 15 mM, more preferably 1 to10 mM, most preferably 5 mM EDTA, wherein further preferably said buffercomprises an electric conductivity below 12 mS/cm, preferably below 10mS/cm, most preferably below 8 mS/cm. In a very preferred embodimentsaid alkaline loading buffer comprises 50 mM Tric-HCl and 5 mM EDTA,wherein the pH at 22° C. is 7.4 (pH is 8.0 at 4° C.) and the electricconductivity is below 8 mS/cm. In a further preferred embodiment saidexposing of said VLP to oxidative conditions is performed by contactingsaid VLP with oxidising compounds, wherein preferably said oxidisingcompounds are selected from the group consisting of: (a) oxidizedglutathione (GSSG), (b) peroxides, preferably H₂O₂, and (c) metal ions,preferably Cu⁺⁺.

The chromatographies of the invention, preferably said first and saidsecond chromatography, are typically and preferably performed incylindrical columns packed with a chromatography matrix, preferably ahydroxyapatite matrix or an anion exchange matrix. Typically andpreferably the chromatographies of the invention are performed usingbuffers referred to as equilibration buffer, washing buffer and elutionbuffer, wherein first equilibration buffer, first washing buffer andfirst elution buffer refer to the buffers used for said firstchromatography and second equilibration buffer, second washing bufferand second elution buffer refer to the buffers used for said secondchromatography. In a preferred embodiment, said chromatography matrix ofsaid first chromatography and/or said hydroxyapatite matrix of saidsecond chromatography, is equilibrated with said first/secondequilibration buffer prior to said binding of said VLP to saidchromatography matrix or said hydroxyapatite matrix. During saidchromatographies said buffers are flowing through said chromatographymatrix in a laminar flow, wherein preferably said flow is driven bygravitation, suction or pressure, preferably by pressure. However, anyother technical setup known in the art and allowing to carry outchromatography may be equally useful.

The buffers used for the chromatographies of the invention, preferablysaid equilibration buffer, said washing buffer and said elution bufferof said first and/or second chromatography, typically and preferablycomprise a pH which is about neutral, more preferably said pH isselected from 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6and 7.8, still more preferably said pH is 7.0 to 7.4, still morepreferably said pH is 7.1 to 7.3, most preferably said pH is 7.2. The pHof the buffers used for the chromatographies of the invention may bestabilized with any buffer system known in the art, preferably by abuffer system commonly used in biochemistry. Preferred buffer systemscomprise a compound selected from the group consisting of: (a) HEPES(N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), (b) MES(2-(N-Morpholino)ethanesulfonic acid), (c) MOPS(3-(N-Morpholino)propanesulfonic acid), (d) TAPS (N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid), (e) TRIS(tris(Hydroxymethyl)aminomethane, Tromethamine), (f) Bis-Tris(bis(2-Hydroxyethyl)amino-tris(hydroxymethyl)methane), (g) Bis-TrisPropane (1,3-bis(tris[Hydroxymethylmethylamino) propane), and (h) anycombination and/or any derivative thereof. Any salt form of thesecompounds may be equally useful. Also inorganic buffer systems such asphosphate buffer or carbonate buffer may be useful for the invention. Ina preferred embodiment the buffers used for the chromatographies of theinvention, preferably said equilibration buffer, said washing buffer andsaid elution buffer of said first and/or second chromatography, comprisea phosphate buffer, preferably potassium or sodium phosphate buffer,most preferably sodium phosphate buffer. Phosphate buffer is a solutioncomprising a mixture of hydrogenphosphate and dihydrogenphosphate,wherein the pH of the solution is determined by the molar ratio of bothcomponents. The preparation of phosphate buffer is within the skill ofthe artisan. Furthermore, the artisan is aware that the presence ofother salts may influence the pH of a phosphate buffer. In particular,the ratio of the salt components of a phosphate buffer needs to beadapted to the presence of potassium or sodium chloride to stabilise acertain pH. In a preferred embodiment the buffers used for thechromatographies of the invention, preferably said equilibration buffer,said washing buffer and said elution buffer of said first and/or secondchromatography, comprise 1 to 100 mM, preferably 5 to 50 mM, morepreferably 10 to 30 mM, still more preferably about 20 mM phosphatebuffer, most preferably 20 mM phosphate buffer. In a very preferredembodiment said buffers comprise 20 mM sodium phosphate buffer.

Chromatography matrices useful in the process of the invention arematerials capable of binding biochemical compounds, preferably proteins,nucleic acids, and/or endotoxins, wherein the affinity of saidbiochemical compounds to said chromatography matrix is influenced by theion composition of the surrounding solution (buffer). Controlling theion composition of said solution allows to use the chromatographymaterials of the invention either in subtractive mode (VLP passesthrough said chromatography matrix, at least certain contaminants bindto said chromatography matrix) or, preferably, in adsorptive mode (VLPbinds to the chromatography matrix). Typically and preferably said VLPbinds to said chromatography matrix, preferably to said first and/or tosaid second chromatography matrix, in the presence of at most about 400mM, more preferably at most about 300 mM, most preferably 100 to 300 mMof said inorganic salt, wherein preferably said inorganic salt ispotassium chloride or sodium chloride, most preferably sodium chloride.Preferred chromatography matrices of the invention are materials whichare capable of reversibly binding said VLP. In one embodiment of theinvention said first chromatography matrix and said secondchromatography matrix, are selected from (a) anion exchange matrix, and(b) hydroxyapatite matrix.

In a preferred embodiment said first chromatography matrix and saidsecond chromatography matrix, preferably said first chromatographymatrix, is an anion exchange matrix, wherein preferably said anionexchange matrix comprises a functional anion exchange group, whereinfurther preferably said functional anion exchange group is a substitutedamine, wherein still further preferably said substituted amine isselected from the group consisting of DEAE (diethylaminoethyl), DMAE(dimethylaminoethyl), TEAE (triethylaminoethyl), TMAE(trimethylaminoethyl), QAE (quaternary amino ethyl), QA (quaternaryamine), and AE (aminoethyl). In very preferred embodiment saidsubstituted amine is a quaternary amine group, wherein said quaternaryamine group is selected from the group consisting of TEAE, TMAE, QAE,and QA. Most preferably said quaternary amine group is TMAE(trimethylaminoethyl).

In a preferred embodiment said first chromatography matrix and saidsecond chromatography matrix, preferably said first chromatographymatrix, is an anion exchange matrix, preferably selected from the groupconsisting of: (a) MATREX® Silica PEI high performance anion exchanger,(b) POROS® HQ, (c) a monolithic anion exchange matrix, preferably aconvective interaction medium (e.g. CIM®-QA (quarternary amino group,BIA Separations Cat. No. 210.5113) or CIM-DEAE (diethylamine, BIASeparations Cat. No. 210.5114), and, very preferably, (d) a tentacleanion exchange matrix.

In a preferred embodiment said first chromatography matrix and saidsecond chromatography matrix, preferably said first chromatographymatrix, is a tentacle anion exchange matrix, preferably comprising resinparticles comprising, preferably on their surface, spacers formed bylinear polymer chains (tentacles), wherein said tentacles aresubstituted with functional groups having anion exchange activity. In apreferred embodiment said resin particles are particles of methacrylateor polyvinylstyrene polymers, preferably comprising a pore size of about800 Å, wherein still more preferably said methacrylate orpolyvinylstyrene polymer are crosslinked. Most preferably said resinparticles consist of crosslinked methacrylate polymer. In a furtherpreferred embodiment said functional group is selected from the groupconsisting of TMAE (Trimethylaminoethyl-), DMAE (Dimethylaminoethyl-),and DEAE (Diethylaminoethyl-). In a very preferred embodiment saidfunctional group is DEAE or TMAE, most preferably TMAE. In a furtherpreferred embodiment said polymer chains forming said tentacles areacrylamide polymers. In a very preferred embodiment said tentacle anionexchange matrix comprises (i) resin particles of methacrylate polymer orof vinyl polymer, preferably of methacrylate polymer, most preferably ofcross-linked methacrylate polymer, (ii) acrylamide tentacles, whereinpreferably said acrylamide tentacles attached to the surface of saidresin particles, and wherein said acrylamide tentacles are substitutedwith TMAE (Trimethylaminoethyl-) groups.

Specifically preferred tentacle ion exchange matrices are FRACTOGEL® EMDTMAE ion exchangers and FRACTOPREP® DEAE ion exchangers (Merck), mostpreferred tentacle ion exchange matrices are FRACTOGEL® ion EMD TMAEexchangers.

Hydroxyapatite matrices are capable of reversibly binding said VLP notonly via their anion exchange activity but also via additionalmechanisms, including for example dipol-dipol interactions, and thusallow very efficient reversible binding of said VLP. Therefore, in afurther preferred embodiment said first chromatography matrix and saidsecond chromatography matrix, preferably said second chromatographymatrix, is a hydroxyapatite matrix, preferably MACRO-PREP® ceramicHydroxyapatite.

In a very preferred embodiment said first chromatography matrix is ahydroxyapatite matrix or an anion exchange matrix, preferably ahydroxyapatite matrix or a tentacle anion exchange matrix, morepreferably a tentacle anion exchange matrix, still more preferably aFRACTOGEL® EMD TMAE ion exchanger or a FRACTOPREP® DEAE ion exchanger,most preferably a FRACTOGEL® ion EMD TMAE exchanger; and said secondchromatography matrix is a hydroxyapatite matrix, preferably MACRO-PREP®ceramic Hydroxyapatite.

FRACTOGEL® ion exchangers are cross-linked porous polymethacrylateresins with pore-sizes of about 800 Å modified according to the tentacletechnology in which functionally substituted acrylamides are grafted tothe surface of the particles. This linking of the functional ionexchanger groups via linear polymer chains renders the ionic groups morereadily accessible for proteins.

FRACTOPREP® ion exchangers are produced employing the same principle asfor FRACTOGEL® ion exchangers but with a vinyl polymer resin baseparticle.

MACRO-PREP® ceramic hydroxyapatite media are a spherical, macroporousform of hydroxyapatite. They are produced by sintering crystallineHydroxyapatite at high temperatures. Type I and II differ in thesintering temperature used for their production which results in adifferent surface composition and different pore sizes of the particles(600-800 Å for Type I, 800-1000 Å for Type II).

MATREX® Silica PEI high performance anion exchange stationary phases arebased on wide pore silica (available with 500 Å and 1,000 Å porediameter).

POROS® HQ is based on a quaternized polyethyleneimine functional groupyielding a high capacity, Perfusion Chromatography® media designed forthe separation and purification of biomolecules.

The main portion of host cell derived impurities remaining in theclarified homogenate of the bacterial host is removed by said purifyingsaid VLP from the clarified homogenate obtained by said clarifying in afirst chromatography, said chromatography comprising the steps of: (i)binding said VLP to a first chromatography matrix, wherein preferablysaid first chromatography matrix is a hydroxyapatite matrix or an anionexchange matrix, more preferably a hydroxyapatite matrix or a tentacleanion exchange matrix, most preferably a tentacle anion exchange matrix,(ii) washing said first chromatography matrix, and (iii) eluting saidVLP from said first chromatography matrix.

In one embodiment said first chromatography matrix is selected from thegroup consisting of: (a) FRACTOGEL® EMD TMAE (M), preferably having aparticle size of 40-90 μm; (b) FRACTOGEL® EMD TMAE Hicap (M), preferablyhaving a particle size of 40-90 μm; (c) FRACTOPREP® DEAE, preferablyhaving a particle size of 30-150 μm; (d) MACRO-PREP® CHT CeramicHydroxyapatite Type I, preferably having a particle size of about 80 μm;(e) MACRO-PREP® CHT Ceramic Hydroxyapatite Type II, preferably having aparticle size of about 80 μm; (f) MATREX® Granular Silica PEI-300 Å,preferably having a particle size of 35-70 μm; (g) MATREX® GranularSilica PEI-1000 Å, preferably having a particle size of 35-70 μm; (h)POROS® 50 HQ, (i) CIM®-QA (quarternary amino group, BIA Separations Cat.No. 210.5113), and (j) CIM®-DEAE.

In one embodiment said first chromatography further comprises the stepof equilibrating said first chromatography matrix with a firstequilibration buffer, wherein said equilibrating is performed prior tosaid binding of said VLP. In a preferred embodiment said firstequilibration buffer comprises an inorganic salt, preferably an alkalinemetal halogenide, more preferably potassium chloride or sodium chloride,most preferably sodium chloride, wherein preferably the concentration ofsaid inorganic salt is chosen to facilitate reversible binding of saidVLP to said first chromatography matrix. In a further preferredembodiment said inorganic salt is an ammonium salt, preferably ammoniumsulphate or ammonium acetate. It is apparent for the artisan that a saltconcentrations above a certain threshold will lead to incomplete bindingof the VLP and, consequently, to loss of VLP. In a very preferredembodiment said first equilibration buffer comprises 50 to 200 mM,preferably 100 to 180 mM, more preferably about 150 mM of potassiumchloride or sodium chloride, preferably sodium chloride. In a furtherpreferred embodiment, said first equilibration buffer comprises at mostabout 200, more preferably at most about 150 mM sodium chloride. In afurther preferred embodiment said first equilibration buffer comprises apH which is about neutral, preferably said pH is 7.2. In a verypreferred embodiment said first chromatography matrix is FRACTOGEL® EMDTMAE and said first equilibration buffer is PBS buffer (Example 4).

In one embodiment said binding of said VLP to said first chromatographymatrix is performed by passing said clarified homogenate containing saidVLP through said first chromatography matrix and, optionally, removingunbound material by further passing equilibration buffer through saidchromatography matrix, wherein preferably the volume of saidequilibration buffer is 1 to 5 times, more preferably 2 to 4 times, mostpreferably 3 times the volume of said first chromatography matrix.Alternatively, said removing of unbound material is performed by furtherpassing a buffer through said chromatography matrix, wherein said bufferhas the same composition as the buffer used for said exposing said VLPto oxidative conditions, and wherein preferably the volume of saidbuffer is 1 to 5 times, more preferably 2 to 4 times, most preferably 3times the volume of said first chromatography matrix.

In one embodiment said first chromatography comprises the step ofwashing said first chromatography matrix, wherein said washing isperformed with said first washing buffer. In a preferred embodiment saidfirst washing buffer comprises an inorganic salt, preferably ahalogenide of an alkaline metal, more preferably sodium chloride orpotassium chloride, most preferably sodium chloride. In a preferredembodiment said first washing buffer comprises the same inorganic saltas said first equilibration buffer, wherein preferably said inorganicsalt is sodium chloride or potassium chloride, most preferably sodiumchloride. The purity of the eluted VLP will depend on the saltconcentration of said first washing buffer. Washing buffer comprising ahigher salt concentration will remove contaminating compounds moreefficiently than a washing buffer comprising a lower salt concentration,whereas a washing buffer comprising a salt concentration above a certainthreshold may cause loss of bound VLP.

In a preferred embodiment said washing of said first chromatographymatrix, is performed with a first washing buffer comprising 300 to 500mM, 350 to 480 mM, 380 to 450 mM, 400 to 440 mM, or 410 to 430 mM of aninorganic salt, preferably of sodium chloride or potassium chloride,most preferably of sodium chloride. In a very preferred embodiment saidfirst washing buffer comprises about 425 mM of said inorganic salt,preferably sodium chloride or potassium chloride, most preferably sodiumchloride, wherein further preferably said first chromatography matrix isan anion exchange matrix, more preferably a tentacle anion exchangematrix, most preferably FRACTOGEL® EMD TMAE. In a still more preferredembodiment said first washing buffer is PBS425 (Example 4). Typicallyand preferably, said washing of said first chromatography matrix isperformed with a volume of said first washing buffer which equals 1 to10 times, preferably 3 to 8 times most preferably about 5 times thevolume of said first chromatography matrix. Alternatively, said washingof said first chromatography matrix is continued until the concentrationof protein as detected by UV absorption at 280 and 300 nm of saidwashing buffer after passing through said chromatography matrix is belowa desired threshold.

In one embodiment said first chromatography comprises the step ofeluting said VLP from said first chromatography matrix, wherein saideluting is performed with a first elution buffer comprising an inorganicsalt, preferably an alkaline metal halogenide, more preferably potassiumchloride or sodium chloride, most preferably sodium chloride. In apreferred embodiment said first elution buffer comprises the sameinorganic salt as said first equilibration buffer and/or said firstwashing buffer. The purity of the eluted VLP will depend on the saltconcentration of the elution buffer. Elution buffer comprising highersalt concentrations may elute more contaminating compounds than thosecomprising lower salt concentrations, whereas elution buffers comprisingsalt at a concentration below a certain threshold may cause loss VLP dueto insufficient elution and result in large elution volumes. For thepurpose of efficient up-scaling of the process, efficient elution of theVLP in minimal elution volumes is desired. Therefore, in a preferredembodiment said eluting of said VLP from said first chromatographymatrix is performed with a first elution buffer comprising aconcentration of said inorganic salt of at least about 480, 490, 400,510, 520, 530, 540, 550, 560, 570, 580, 590 or 600 mM, wherein saidinorganic salt is preferably a halogenide of an alkaline metal, morepreferably sodium chloride or potassium chloride, most preferably sodiumchloride. In a very preferred embodiment said eluting is performed witha first elution buffer comprising 500 to 600 mM, preferably 520 to 580mM, more preferably 530 to 570 mM, still more preferably 540 to 560 mM,most preferably about 550 mM of said inorganic salt, preferably ofsodium chloride or potassium chloride, most preferably of sodiumchloride. In a very preferred embodiment said first elution buffer isPBS550 (Example 4). In another embodiment eluting of said VLP from saidfirst chromatography matrix is performed with a concentration gradientof said inorganic salt.

Depending on the specific biochemical features of the VLP and of saidfirst chromatography matrix the separation of the VLP from contaminatingcompounds may be improved by application of linear or non-linearconcentration gradients. In a preferred embodiment said eluting of saidVLP, preferably of a VLP of RNA bacteriophage Qβ, from said firstchromatography matrix is performed with a linear gradient of saidinorganic salt in said first elution buffer, wherein said lineargradient preferably ranges from 300 to 900 mM, more preferably from 400to 700 mM, most preferably from 425 to 650 mM; and wherein furtherpreferably the volume of said first elution buffer is 1 to 5 times,preferably 2 to 4 times, most preferably 3 times the volume of saidchromatography matrix; and wherein still further preferably saidinorganic salt is sodium chloride or potassium chloride, most preferablysodium chloride; and wherein still further preferably said firstchromatography matrix is an anion exchange matrix, more preferably atentacle anion exchange matrix, most preferably FRACTOGEL® EMD TMAE.

In a further preferred embodiment said first chromatography matrix is ahydroxyapatite matrix, preferably hydroxyapatite matrix comprising apore size of 800-1000 Å, and said eluting is performed using aco-gradient of a hydrogenphosphate/dihydrogenphosphate and an inorganicsalt, wherein said inorganic salt preferably is a halogenide of analkaline metal, more preferably a chloride, still more preferablypotassium chloride or sodium chloride, most preferably sodium chloride.In a very preferred embodiment said said eluting is performed by aco-gradient of sodium hydrogenphosphate/dihydrogenphosphate and sodiumchloride.

The elution profile of the VLP from the anion exchange matrix can bemonitored by registration of the UV absorption, wherein registration ofthe UV absorption at 280 and 300 nm is especially useful for thedetection of VLPs of RNA bacteriophages, preferably of VLPs of RNAbacteriophage Qβ, The artisan is able to interpret such elution profilesand to identify the fractions containing the purified VLP. For example,in case said first chromatography matrix is an anion exchange matrix andsaid first elution buffer comprises 550 mM sodium chloride, the VLP istypically contained in the fraction from about 0.5 to about 2 times thevolume of the anion exchange matrix. In case said first chromatographymatrix is an anion exchange matrix and said first elution buffercomprises a gradient of 425 to 650 mM sodium chloride in three times thevolume of said anion exchange matrix, the VLP is typically contained inthe fraction from about 1.3 to about 2.6 times the volume of the anionexchange matrix. In one embodiment said purifying of said VLP from saidclarified homogenate further comprises the step of obtaining thefraction of the eluate of said first chromatography containing said VLP.In a further embodiment said purifying of said VLP from said clarifiedhomogenate further comprises the step of diluting said fraction in sucha way that the concentration of said inorganic salt in said fraction isin a range which allows binding of said VLP to said secondchromatography matrix. Typically and preferably, said fraction isdiluted in a ratio of about 1:2 with a buffer which is essentially freeof said inorganic salt. In a preferred embodiment, said fraction isdiluted in such a way that the concentration of said inorganic salt is100 to 400 mM, preferably 200 to 400 mM, more preferably 200 to 300 mM,most preferably about 250 mM. In a further embodiment said purifying ofsaid VLP from said clarified homogenate further comprises the optionalstep of sterile filtering the eluate of said first chromatography, saidfraction containing said VLP, or said diluted fraction through a sterilefilter having a pore size of about 0.18 to 0.25 μm, preferably 0.20 to0.22 μm, most preferably 0.22 μm.

The process of the invention further comprises a second chromatography,wherein said second chromatography is particularly efficient in removingendotoxin contaminations. In one embodiment said further purifying saidVLP in a second chromatography from the eluate obtained by said firstchromatography; or, preferably, from said fraction containing said VLPor from the filtrate obtained by said optional sterile filtering; saidsecond chromatography comprising the steps of: (i) binding said VLP to asecond chromatography matrix, wherein preferably said secondchromatography matrix is a hydroxyapatite matrix; (ii) washing saidsecond chromatography matrix; and (iii) eluting said VLP from saidsecond chromatography matrix.

In a preferred embodiment said second chromatography matrix is ahydroxyapatite matrix, wherein preferably said hydroxyapatite matrix isa ceramic hydroxyapatite matrix, wherein preferably said ceramichydroxyapatite matrix comprises a particle size of about 80 μm and apore size of the particles of about 800-1000 Å. In a very preferredembodiment said hydroxyapatite matrix is a MACRO-PREP® CHT CeramicHydroxyapatite matrix. In a still more preferred embodiment saidhydroxyapatite matrix is MACRO-PREP® CHT Ceramic Hydroxyapatite Typ II.

In a further preferred embodiment said second chromatography comprisesthe step of equilibrating said second chromatography matrix with asecond equilibration buffer, wherein said second equilibration bufferpreferably comprises an inorganic salt, wherein further preferably saidsecond equilibration buffer comprises said inorganic salt in aconcentration which facilitates binding of said VLP to said secondchromatography matrix, wherein further preferably said secondchromatography matrix is a hydroxyapatite matrix. In a preferredembodiment said concentration of said inorganic salt in said secondequilibration buffer is at most 400 mM, preferably at most 300 mM.

It was found that binding of the VLP to a hydroxyapatite matrix in theabsence of an inorganic salt is instable and causes loss of VLP duringthe following washing step, while salt concentrations above a certainthreshold will lead to incomplete binding. In a preferred embodimentsaid second equilibration buffer comprises the same inorganic salt assaid first equilibration buffer and/or said first washing buffer and/orsaid first elution buffer. In a further preferred embodiment said secondchromatography matrix is a hydroxyapatite matrix and said secondequilibration buffer comprises 50 to 200 mM, preferably 100 to 180 mM,more preferably about 150 mM of potassium chloride or sodium chloride,preferably sodium chloride. In a very preferred embodiment said secondequilibration buffer is PBS (Example 5).

In a preferred embodiment said binding of said VLP to said secondchromatography matrix, preferably to said hydroxyapatite matrix, isperformed in the presence of 100 to 400 mM, preferably 200 to 400 mM,more preferably 200 to 300 mM, most preferably about 250 mM of saidinorganic salt, wherein said inorganic salt preferably is potassiumchloride or sodium chloride, most preferably sodium chloride.

In a further embodiment said washing of said second chromatographymatrix is performed with a second washing buffer, wherein preferablysaid second washing buffer comprises an inorganic salt, preferably ahalogenide of an alkaline metal, more preferably sodium chloride orpotassium chloride, most preferably sodium chloride. In a preferredembodiment said second washing buffer comprises the same inorganic saltas said second equilibration buffer, preferably sodium chloride orpotassium chloride, most preferably sodium chloride. The purity of theeluted VLP will depend on the salt concentration of the second washingbuffer. Washing buffers comprising higher salt concentrations willremove contaminating compounds more efficiently than those comprisinglower salt concentrations, whereas washing buffers comprising salt at aconcentration beyond a certain threshold may cause loss of bound VLP. Ina preferred embodiment said second washing buffer comprises 50 to 400mM, preferably 100 to 300 mM, more preferably 120 to 200 mM, still morepreferably 130 to 170 mM, still more preferably 140 to 160 mM, mostpreferably 145 to 155 mM of said inorganic salt, preferably of sodiumchloride or potassium chloride, most preferably sodium chloride. In avery preferred embodiment said second washing buffer comprises 150 mM ofsaid inorganic salt, preferably of sodium chloride or potassiumchloride, most preferably sodium chloride. In a still more preferredembodiment said second washing buffer is PBS (Example 5). In a furtherpreferred embodiment said washing of said second chromatography matrix,is performed by passing said second washing buffer through said secondchromatography matrix, wherein the volume of said second washing bufferpassed through said second chromatography matrix preferably is 1 to 10times, more preferably 3 to 8 times, most preferably 5 times the volumeof said second chromatography matrix.

In a further embodiment said second chromatography comprises the step ofeluting said VLP from said second chromatography matrix, wherein saideluting is performed by a second elution buffer, wherein said secondelution buffer preferably comprises an inorganic salt, wherein furtherpreferably said inorganic salt is an alkaline metal halogenide, morepreferably potassium chloride or sodium chloride, most preferably sodiumchloride. In a further preferred embodiment said second elution buffercomprises an inorganic salt, wherein preferably said inorganic salt isthe same salt as contained in said second equilibration buffer and/orsaid second washing buffer. In a further embodiment said eluting of saidVLP from said second chromatography matrix is performed with an elutionbuffer comprising a concentration of said inorganic salt of at leastabout 900, 1000, 1200, 1500, 2000 or 3000 mM, wherein said inorganicsalt is preferably a halogenide of an alkaline metal, more preferablysodium chloride or potassium chloride, most preferably sodium chloride.

In a further preferred embodiment said second elution buffer comprises acombination of phosphorous salts (phosphate buffer), preferablyhydrogenphosphate and dihydrogenphosphate of potassium or sodium,preferably of sodium. In a preferred embodiment said second elutionbuffer comprises 200 to 500 mM, more preferably 200 to 400 mM, mostpreferably about 300 mM phosphate buffer, preferably sodium phosphatebuffer.

A second elution buffer only containing said alkaline metal halogenideeither requires a high concentration of said alkaline metal halogenidewhich may lead to the precipitation of said VLP or, at lower saltconcentrations, leads to high elution volumes which are undesired withrespect to the up-scale of the process. A second elution buffer which isessentially free of said alkaline metal halogenide and only based onsaid combination of phosphorous salts would, on the other hand, alsoresult in high eluate volumes. It was found that second elution bufferscomprising a combination of both, said alkaline metal halogenide andsaid combination of phosphorous salts (phosphate buffer), allow forefficient elution of said VLP, for minimizing the elution volume, forminimizing the total salt concentration in the eluate, and, thus, forefficient up-scaling of the process. In a further preferred embodimentsaid second elution buffer therefore comprises a mixture of saidalkaline halogenide and of said combination of phosphorous salts,wherein preferably the concentration of said combination of phosphoroussalts is the lowest concentration allowing elution of said VLP when saidcombination of phosphorous salts is used alone, i.e. in the absence ofsaid alkaline metal halogenide. In a very preferred embodiment saidsecond elution buffer comprises an alkaline metal halogenide, preferablypotassium chloride or sodium chloride, most preferably sodium chloride;and a combination of phosphorous salts, preferably potassium or sodiumhydrogenphosphate/dihydrogenphosphate, most preferably sodiumhydrogenphosphate/dihydrogenphosphate, wherein preferably theconcentration of said alkaline metal halogenide is 900 to 1200 mM, morepreferably about 900 mM; and wherein further preferably theconcentration of said combination of phosphorous salts is 200 to 300 mM,most preferably about 200 mM; and wherein still further preferably saidVLP is a VLP of RNA bacteriophage Qβ. In a very preferred embodimentsaid second elution buffer is HSB and sai VLP preferably is a VLP of RNAbacteriophage Qβ (Example 5).

In a further preferred embodiment said second elution buffer comprisesan alkaline metal halogenide, preferably potassium chloride or sodiumchloride, most preferably sodium chloride; and a combination ofphosphorous salts, preferably potassium or sodiumhydrogenphosphate/dihydrogenphosphate, most preferably sodiumhydrogenphosphate/dihydrogenphosphate, wherein preferably theconcentration of said alkaline metal halogenide is 50 to 500 mM, morepreferably about 250 mM; and wherein further preferably theconcentration of said combination of phosphorous salts is 10 to 200 mM,most preferably about 50 mM; and wherein still further preferably saidVLP is a VLP of RNA bacteriophage AP205.

In a further preferred embodiment said eluting of said VLP from saidsecond chromatography matrix is performed with a concentration gradientof said inorganic salt in said second elution buffer. Depending on thespecific features of the VLP and the hydroxyapatite matrix theseparation of the VLP from contaminating compounds may be improved byapplication of linear or non-linear concentration gradients. Forexample, the VLP may be eluted with a concentration gradient of saidalkaline metal halogenide from 900 to 3000 mM and/or a concentrationgradient of said phosphorous salt from 100 to 400 mM.

The elution profile of said VLP from said second chromatography matrixcan be monitored by registration of the UV absorption, whereinregistration of the UV absorption at 280 and 300 nm is especially usefulfor the detection of VLPs of RNA bacteriophages, preferably of VLPs ofRNA bacteriophage Qβ. The artisan is able to interpret such elutionprofiles and to identify the fractions containing the purified VLP. Asan example, in case said second chromatography matrix is hydroxyapatiteand said second elution buffer comprises 900 mM sodium chloride and 200mM sodium hydrogenphosphate/dihydrogenphosaphate, said VLP is typicallycontained in the fraction from about 0.5 to about 3 times the volume ofsaid second chromatography matrix.

The purity of the VLP preparation may be assessed by analytical sizeexclusion chromatography (Example 7). Remaining contamination of the VLPpreparation with host cell protein may also be detected by immunologicalmeans, for example by a ELISA detecting E. coli derived protein (e.g. E.coli HCP ELISA kit, Cygnus Technologies Inc., cat. nos. F010 and F410).The VLP contained in the eluate of said second chromatography may bedesalted and, if required, finally purified in a so called “polishingstep”. In a further preferred embodiment the process of the inventioncomprises the step of finally purifying said VLP, wherein preferablysaid finally purifying of said VLP is performed by precipitating saidVLP, preferably with ammonium sulphate, and preferably by at least onesubsequent third chromatography, wherein preferably said at least onesubsequent third chromatography is a size exclusion chromatography. In amore preferred embodiment said finally purifying said VLP is performedby at least one third chromatography, wherein preferably said at leastone third chromatography is selected from the group consisting of: (a)immobilised metal ions affinity chromatography (IMAC), preferably IMC insubtractive mode (contaminants selectively bind to the chromatographymatrix); (b) hydrophobic interaction chromatography (HIC), wherein saidHIC may be performed in subtractive or in adsorptive mode; (c) membraneadsorption and (d) size exclusion chromatography. In a further preferredembodiment said at least one third chromatography comprises membraneadsorption, wherein preferably said membrane adsorption is performedwith an adsorption membrane selected from the group consisting of: (a)polyethylenimine coated membrane, preferably PALL, Mustang E, Cat. No.CLM05MSTGEP1, CL3MSTGEP1, NP6MSTGEP1, NP7MSTGEP1, or NP8MSTGEP1; and (b)adsorber membranes comprising quarternary amino groups, preferably PallMustang Q, Pall, Cat. No. CLM05MSTGQP1 or Sartorius Sartobind Q-membraneadsorber (Sartorius, Cat. No. Q15X; Q100X). In a further preferredembodiment said at least one third chromatography comprises ahydrophobic interaction chromatography (HIC, Example 14) followed by asize exclusion chromatography. In a further preferred embodiment said atleast one third chromatography comprises a immobilised metal ionsaffinity chromatography (IMAC, Example 15), wherein preferably saidmetal ions are Zn⁺⁺ ions or Cu⁺⁺ ions, and wherein further preferablysaid IMAC is performed in subtractive mode (contaminants bind tochromatography matrix); wherein further preferably said IMAC is followedby size exclusion chromatography.

Size exclusion chromatography is useful for further purifying and/orre-buffering said VLP. In a very preferred embodiment said at least onethird chromatography is at least one, preferably exactly one, sizeexclusion chromatography. In a preferred embodiment said size exclusionchromatography comprises the steps of loading said fraction of theeluate of said second chromatography to a gel filtration matrix, whereinsaid gel filtration matrix preferably is equilibrated with a bufferhaving a composition which is desired for storage or further processingof said VLP. In a preferred embodiment said buffer comprises aninorganic salt, preferably a halogenide of an alkaline metal, morepreferably potassium chloride or sodium chloride, most preferably sodiumchloride, wherein the concentration of said inorganic salt is about 50to 500 mM, preferably 100 to 300, most preferably about 150 mM.

In case no remaining contaminants are detectable in the preparation ofsaid VLP, e.g. the elution profile of said analytical size exclusionchromatography does not reveal extra peaks, said gel filtration matrixpreferably is a desalting matrix. In a preferred embodiment said gelfiltration matrix is a desalting matrix, wherein said desalting matrixpreferably is a SEPHADEX® matrix, most preferably SEPHADEX® G-25.

In case the VLP preparation still comprises contaminants as, for exampledetected by analytical size exclusion chromatography, ELISA (e.g. E.coli HCP ELISA kit, Cygnus Technologies Inc., cat. nos. F010 and F410)or any other assay, said gel filtration matrix preferably is a matrixhaving a separation characteristic within the range of 2×10⁴ to 2×10⁷Dalton. In a preferred embodiment said gel filtration matrix is a gelfiltration matrix having a separation characteristic within the range of2×10⁴ to 2×10⁷ Dalton. In a further preferred embodiment said gelfiltration matrix is selected from the group consisting of: (a)SEPHAROSE® 4 FF, (b) SEPHACRYL®-S500 HR, (c) SEPHACRYL®-S1000 SF, (d)Toyopearl HW-65F, (e) SEPHAROSE® CL-4B and (f) SEPHACRYL®-S400. Mostpreferably, said gel filtration matrix is SEPHAROSE® CL-4B. Said sizeexclusion chromatography further comprises the step of eluting the VLPfrom said gel filtration matrix by isocratic elution, i.e. the elutionbuffer has about the same, preferably the same composition as the bufferused for equilibration. The UV absorption of the flow through isrecorded, preferably at 280 nm and 300 nm, the fraction containing theVLP is collected.

In a further embodiment the process further comprises the step ofsterile filtering said VLP, wherein the fraction containing the VLP ofthe eluate of said at least one third chromatography, preferably of saidsize exclusion chromatography, is filtrated through a sterile filter,preferably having a pore size of about 0.18 to 0.25 μm, preferably 0.20to 0.22 μm, most preferably 0.22 μm.

In a further embodiment said finally purifying said VLP by said at leastone third chromatography comprises the step of concentrating thesolution comprising said VLP by filtration, preferably by tangentialflow filtration, most preferably by tangential flow filtration using aBiomax 100 membrane, wherein preferably said concentrating is performedprior to the last of said at least one third chromatography, whereinfurther preferably the last of said at least one third chromatography isa size exclusion chromatography.

The purified VLP may by stored at −75±15° C. until further processing.

As the process of the invention is a process for the purification ofself-assembled VLP, it is neither possible nor intended to remove hostcell derived nucleic acids and host cell derived proteins which areencapsulated inside the VLP using the process of the invention.Encapsulated host cell derived nucleic acids and host cell derivedproteins often are an essential stabilizing element of the VLP andconstitute an integral element of the VLP. They are therefore notregarded as impurities in the context of this invention. Recombinantlyexpressed self-assembled VLPs contain host cell derived nucleic acidsand host cell derived protein in a rather constant ratio relative to theamount of capsid protein forming the VLP, wherein this ratio may differbetween different VLP species. As an example, purified recombinantlyexpressed and self-assembled VLP of RNA bacteriophage Qβ typicallycontains per 100 μg Qβ capsid protein about 25 to 35 μg RNA, about 4 to6 ng host cell DNA and about 2 μg host cell protein.

A very preferred embodiment of the invention is a process for thepurification of a VLP of bacteriophage Qβ from a recombinant bacterialhost expressing said VLP, the process comprising the steps of: (a)homogenizing said bacterial host; (b) clarifying the homogenate obtainedby said homogenizing; (c) purifying said VLP from the clarifiedhomogenate obtained by said clarifying in a first chromatographycomprising the steps of: (i) binding said VLP to a FRACTOGEL® EMD TMAE(M) matrix in the presence of at most 150 mM sodium chloride; (ii)washing said FRACTOGEL® EMD TMAE (M) matrix in the presence of about 425mM sodium chloride; and (iii) eluting said VLP from said FRACTOGEL® EMDTMAE (M) matrix in the presence of about 550 mM sodium chloride; whereinsaid first chromatography is performed at a pH of 7.2; (d) furtherpurifying said VLP from the eluate obtained by said first chromatographyin a second chromatography comprising the steps of: (i) binding said VLPto a MACRO-PREP® CHT Ceramic Hydroxyapatite Type II matrix in thepresence of about 250 mM sodium chloride; (ii) washing said MACRO-PREP®CHT Ceramic Hydroxyapatite Type II matrix in the presence of about 150mM sodium chloride; (iii) eluting said VLP from said MACRO-PREP® CHTCeramic Hydroxyapatite Type II matrix in the presence of about 900 mMsodium chloride and about 200 mM sodium phosphate buffer; wherein saidsecond chromatography is performed at a pH of about 7.2; and (e) finallypurifying said VLP contained in the eluate of said second chromatographyby at least one third chromatography, wherein said at least one thirdchromatography is exactly one size exclusion chromatography, whereinsaid size exclusion chromatography is performed in the presence of about150 mM sodium chloride, and wherein further said size exclusionchromatography is performed using a gel filtration matrix, wherein saidgel filtration matrix is SEPHAROSE® CL-4B; wherein said steps areperformed in the given order.

A further preferred embodiment of the invention is a process for thepurification of a VLP of bacteriophage AP205 from a recombinantbacterial host expressing said VLP, the process comprising the steps of(a) homogenizing said bacterial host; (b) clarifying the homogenateobtained by said homogenizing; (c) purifying said VLP from the clarifiedhomogenate obtained by said clarifying in a first chromatographycomprising the steps of: (i) binding said VLP to a FRACTOGEL® EMD TMAE(M) matrix in the presence of at most 150 mM sodium chloride; (ii)washing said FRACTOGEL® EMD TMAE (M) matrix in the presence of about 425mM sodium chloride; and (iii) eluting said VLP from said FRACTOGEL® EMDTMAE (M) matrix in the presence of about 550 mM sodium chloride; whereinsaid first chromatography is performed at a pH of 7.2; (d) furtherpurifying said VLP from the eluate obtained by said first chromatographyin a second chromatography comprising the steps of: (i) binding said VLPto a MACRO-PREP® CHT Ceramic Hydroxyapatite Type II matrix in thepresence of about 100 mM sodium chloride and at most about 5 mM sodiumphosphate buffer; (ii) washing said MACRO-PREP® CHT CeramicHydroxyapatite Type II matrix in the presence of about 100 mM sodiumchloride and at most about 5 mM sodium phosphate buffer; (iii) elutingsaid VLP from said MACRO-PREP® CHT Ceramic Hydroxyapatite Type II matrixin the presence of about 250 mM sodium chloride and about 50 mM sodiumphosphate buffer; wherein said second chromatography is performed at apH of about 7.2; and (e) finally purifying said VLP contained in theeluate of said second chromatography by at least one thirdchromatography, wherein said at least one third chromatography isexactly one size exclusion chromatography, wherein said size exclusionchromatography is performed in the presence of about 150 mM sodiumchloride, and wherein further said size exclusion chromatography isperformed using a gel filtration matrix, wherein said gel filtrationmatrix is SEPHAROSE® CL-4B; wherein said steps are performed in thegiven order.

EXAMPLES Example 1

Cell Disruption

Solutions used for this process step were composed as described in Table1.

TABLE 1 Composition of process solutions for cell disruption. EB⁻-buffer43.89 mM Tris•HCl 6.11 mM Tris Base 5.0 mM EDTA Na3•3 H2O 10% Triton ®X-100 10% (v/v) Triton ® X-100 saturated Tris Base 500 g/l Tris Base

Cell disruption of E. coli cells expressing Qβ was performed as follows:Bacterial cell pellets retrieved from the −80° C. storage device werethawed by resuspension in 2 ml EB⁻-buffer per 1 g cell pellet at 24° C.The thawed suspension was degassed for 10-15 minutes under vacuum before10% TRITON® X-100 was added to a final concentration of 0.1% (v/v).After stirring for 5 minutes, the cells were disrupted by three passagesat 700±50 bar through an APV LAB 1000 high pressure liquid homogenizer(HPLH). The resulting homogenate was adjusted to pH≧7.8 by the additionof saturated Tris base and diluted 1:2 with EB⁻-buffer.

Example 2

Tangential Flow Filtration (TFF) and Sterile Filtration

A new TFF membrane suitable for the processing of high viscosity media,e.g. PVDF (Pellicon, Millipore), stabilized cellulose (Sartocon,Sartorius) and Polyethersulfon (SUPOR Pall), equipped with a 0.45 μmpore width membrane and an effective membrane area of 0.1 m² forhomogenate derived from 400 g cell wet weight was sanitized andequilibrated with EB⁻-buffer. The feed and retentate outlets of themembrane holder were connected to a container containing dilutedhomogenate derived from 400 g cell wet weight. Diafiltration againstEB⁻-buffer with a feed pressure of 1.0±0.2 bar was performed until twotimes the volume of the diluted homogenate had been collected aspermeate. The permeate was filtrated over a 0.22 μm pore widthsterilizing grade filtering unit and stored at 4° C.

Example 3

Clarification by Centrifugation as Alternative to Tangential FlowFiltration

The homogenate produced in Example 2 was not diluted prior to this step.The homogenate was centrifuged at 4° C. for 105 minutes at 10.000 g. Thesupernatant was decanted from the pellet without transferring the softoverlay and recentrifuged at 4° C. for 60 minutes at 10.000 g. Thesupernatant was decanted from any pellet present, diluted 1:2 withEB⁻-buffer, filtrated over a 0.22 μm pore width sterilizing gradefiltering unit and stored at 4° C.

Example 3a

Clarification by a Combination of Centrifugation and TFF

The homogenate produced in Example 2 was not diluted prior to this step.The homogenate was centrifuged at 4° C. for 120 minutes at 10.000 g. Thesupernatant was decanted from the pellet without transferring the softoverlay and subjected to tangential flow filtration as described inExample 2.

Example 4

Anion Exchange (AIX) Chromatography on FRACTOGEL® EMD TMAE

TABLE 2 Composition of process solutions for AIX chromatography BuffersComponent PBS PBS425 PBS550 Na₂HPO₄•2 H₂O 15.15 mM 16.47 mM 16.98 mMNaH₂PO₄•2 H₂O  4.85 mM  3.53 mM  3.02 mM NaCl   150 mM   425 mM   550 mM

The AIX chromatography was performed as follows: Sterile filtratedcleared cell homogenate produced either by TFF (Example 2) or bycentrifugation (Example 3) was loaded on a FRACTOGEL® EMD TMAE column(bed volume of 350-450 ml for a sample derived from 80 g cell wetweight) equilibrated in PBS buffer. Unbound proteins were washed of thecolumn with about 3 column volumes PBS and weakly bound impurities wereeluted with 5 column volumes PBS425 before Qβ VLP was eluted withPBS550. Qβ VLP of sufficient purity for further processing elutedbetween 0.3 and 0.8 column volumes after the step to PBS550.Alternatively, Qβ VLP was eluted with PBS comprising a gradient of NaClfrom 425 mM to 650 mM in 3 column volumes.

Example 5

Chromatography on Ceramic Hydroxyapatite (cHA)

The cHA chromatography was performed as follows: Peak fractions from theseparation on FRACTOGEL® EMD TMAE were pooled and diluted 1:2 withNaPP-buffer pH 7.0. The diluted sample was filtrated using a 0.22 μmpore width sterilizing grade filter unit and loaded on a MACRO-PREP®ceramic Hydroxyapatite Type II column (bed volume of 125-175 ml forsample derived from 80 g cell wet weight) equilibrated in PBS. Unboundsample was eluted with 5 column volumes PBS before elution of Qβ VLP wasinitiated by a step to HSB. Qβ VLP for further processing eluted between0.6 and 1.8 column volumes after the step to HSB. Qβ VLP containing peakfractions were pooled and stored at 4° C.

TABLE 3 Composition of process solutions for cHA cbromatography BuffersComponent HSB NaPP pH 7.0 PBS Na₂HPO₄•2 H₂O 176.4 mM 10.70 mM  15.15 mMNaH₂PO₄•2 H₂O 23.60 mM 9.30 mM  4.85 mM NaCl   900 mM —   150 mM

Example 6

Screening for Suitable Chromatography Media

The chromatography media listed below were tested for their affinity forQβ VLP.

-   -   FRACTOGEL® EMD TMAE (M) particle size 40-90 μm, Merck,        Darmstadt, Germany (Order No. 1.16881.0500 for 500 ml)    -   FRACTOGEL® EMD TMAE Hicap (M) particle size 40-90 μm, Merck,        Darmstadt, Germany (Order No. 1.10316.0100 for 100 ml)    -   FRACTOPREP® DEAE particle size 30-150 μm, Merck, Darmstadt,        Germany (Order No. 1.17971.0010 for 10 ml)    -   FRACTOPREP® TMAE particle size 30-150 μm, Merck, Darmstadt,        Germany (Order No. 1.17973.0100 for 100 ml)    -   MACRO-PREP® CHT Ceramic Hydroxyapatite Type I, 80 μm particle        size, Bio-Rad Laboratories, Hercules, USA (Order No. 157-0080        for 100 g)    -   MACRO-PREP® CHT Ceramic Hydroxyapatite Type II, 80 μm particle        size, Bio-Rad Laboratories, Hercules, USA (Order No. 157-8000        for 100 g)    -   MATREX® Granular Silica PEI-300 Å particle size 35-70 μm,        Millipore, Bedford, USA (Catalogue Number: 84912 for 100 g)    -   MATREX® Granular Silica PEI-1000 Å particle size 35-70 μm,        Millipore, Bedford, USA (Catalogue Number: 84959 for 100 g)    -   POROS® 50 HQ, PerSeptive Biosystems, Framingham, USA (Order No.        1-2559-03 for 25 ml)    -   Q SEPHAROSE® XL, GE Healthcare, Piscataway, USA (Order No.        17-5072-01 for 300 ml)    -   UNOSPHERE® Q Bio-Rad Laboratories, Hercules, USA (Order No.        156-0101 for 25 ml)

Testing was performed essentially as follows: Qβ VLP solutions fromdifferent process stages containing 0-200 mM NaCl in different buffersystems at pH values between 7.0 and 8.0 were applied on small scalecolumns packed with the respective column matrix and unbound materialwas washed out with running buffer. Bound components from the samplewere eluted either by linear or step gradients during which theconcentrations of either salt or buffer components or both were raised.The observed elution profiles were interpreted in terms of bindingcapacity and selectivity of elution. FRACTOGEL® EMD TMAE (M), FRACTOGEL®EMD TMAE Hicap (M) and MACRO-PREP® CHT Ceramic Hydroxyapatite Type IIcould be identified as chromatography matrices showing a bindingcapacity which is most suitable for a large scale production.

Example 7

Determination of Qβ VLP by Analytical Size Exclusion Chromatography

Analysis of Qβ particles by analytical size exclusion chromatography wasperformed using a TskgelG5000 PWXL-column (10 μm, 7.8×300 mm, TosoHBiosep; Cat.-No. 08023) equilibrated in phosphate buffered saline (20 mMNa₂HPO₄/NaH₄PO₄, 150 mM NaCl pH 7.2). Run conditions for the analysisare summarized in Table 4.

TABLE 4 Run conditions for SE-HPLC analysis of Qβ VLP Flow 0.8 ml/minRunning buffer 20 mM Na₂HPO₄/NaH₂PO₄ 150 mM NaCl pH 7.2 Sampleconcentration 1 mg/ml Injection volume 40 μl Column temperature 25° C.Run time per sample 20 minutes

Purity of Qβ VLP was determined by integration of the peaks in theelution profile at 260 nm.

Example 8

Selection of a Suitable Membrane for Clarification of Cell Homogenatesby TFF

TFF membranes suitable for the processing of high viscosity media withdifferent pore sizes were tested. These membranes included the deviceslisted below:

-   -   Pellicon 2 Mini Filter Module, 0.45 μm-Durapore membrane, screen        V, filter area 0.1 m², Bedford, Mass., USA (Cat. No. P2HVMPV01)    -   Pellicon 2 Mini Ultrafiltration Module, 1000 kD-Biomax membrane,        screen C, filter area 0.1 m², Bedford, Mass., USA (Cat. No.        P2B01MC01)    -   Sartocon Slice Microfiltration Cassette, 0.2 μm-Hydrosart        membrane, open channel, membrane area 0.1 m², Sartorius, Germany        (Cat. No. 305 186 07 01 O-SG)    -   Centramate Tangential Flow Filtration Cassette, 0.45 μm-Supor        membrane, suspended screen, membrane area 0.1 m², Pall, USA        (Order No. PS M45 C11)    -   Ultran-Slice Membrane Cassette, 0.2 μm-PES membrane, open        channel, membrane area 0.1 m², Schleicher & Schuell, Germany        (Order No. 10478685).

Homogenate prepared according to Example 1 (with and without the final1:2 dilution step) was diafiltrated over the respective membrane againstEB⁻-buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA). The achievable permeateflow across the membrane was investigated in addition to proteinconcentration and relative content of Qβ VLP in the recovered permeate.Pore sizes smaller than 0.22 μm led to effective retention of Qβ VLPwhile pore sizes larger than 0.45 μm were assumed to severely impedesterile filtration of the permeate solution. Best results could beobtained with 0.45 μm pore size.

Example 9

Re-Buffering by Size Exclusion Chromatography

The size exclusion chromatography is performed as follows: Pooled peakfractions from the separation on MACRO-PREP® ceramic Hydroxyapatite TypeII are loaded on a SEPHAROSE® CL-4B column (bed volume of 1500-1750 mlfor sample derived from 80 g cell wet weight, bed height 45-75 cm)equilibrated in PBS buffer (20 mM Na₂HPO₄/NaH₂PO₄, 150 mM NaCl pH 7.2).Elution is achieved by isocratic elution with PBS buffer. Symmetricalfractionation of the Qβ VLP main peak is initiated 0.475 column volumesafter start of the loading procedure. The desalted Qβ VLP solution isfiltrated via a 0.22 μm pore width sterilizing grade filter unit,aliquoted and stored at −80° C.

Example 10

Analysis of Endotoxin Content in Qβ VLP Solutions

Testing for endotoxin contamination of Qβ VLP containing solutions wasperformed as laid out in Pharm Eur 2.6.14. Method E using eitherBiowhittaker Kinetic-QCL® Kinetic Chromogenic Assay or Charles RiverEndochrome-K™ kits. Results usually obtained with the purificationprocedure presented here are in the range of 0.5-5 EU/100 μg Qβ VLP.

Example 11

Purification of AP205 VLP

AP205 VLP is purified from bacteria expressing AP205 capsid proteinfollowing the procedure of Examples 1, 3, 4, and 9. The endotoxincontent of the preparation is between 0.5-5 EU/100 μg AP205 VLP asdetermined according to Pharm Eur 2.6.14. Method E using eitherBiowhittaker Kinetic-QCL® Kinetic Chromogenic Assay or Charles RiverEndochrome-K™ kits.

Example 12

Purification of HBc VLP

HBc VLP is purified from bacteria expressing the protein of SEQ ID NO:1or SEQ ID NO:2, essentially following the procedure of Examples 1, 3, 4,and 9. The endotoxin content of the preparation is between 0.5-5 EU/100μg HBc VLP as determined according to Pharm Eur 2.6.14. Method E usingeither Biowhittaker Kinetic-QCL® Kinetic Chromogenic Assay or CharlesRiver Endochrome-K™ kits.

Example 13

Oxidation of Qβ VLP

The oxidation step was performed as follows: Sterile filtrated clearedcell homogenate produced either by TFF (Example 2) or by centrifugation(Example 3) or a combination thereof was stirred for 12 hours in acontainer that allowed free circulation of air to the surface of thesolution. Stirring was performed in way that allowed maximum dissolutionof oxygen in the solution without causing foaming. The mature Qβ VLPsolution, containing an appropriate amount of disulfide bonds, isfiltrated via a 0.22 μm pore width sterilizing grade filter unit.

Example 14

Chromatography on Hydrophobic Interaction Chromatography Matrices

Chromatography on Phenyl SEPHAROSE® High Performance was performed asfollows: A peak fraction from the separation on FRACTOGEL® EMD TMAE wasadjusted to 1.0 M (NH₄)₂SO₄ in 20 mM sodium phosphate buffer pH 7.2 andloaded on a Phenyl SEPHAROSE® High Performance column (10 mg VLP/ml bedvolume) equilibrated in NaPP-buffer pH 7.2 containing 1.0 M (NH₄)₂SO₄.Unbound sample was eluted with 5 column volumes equilibration bufferbefore elution of Qβ VLP was initiated by a linear gradient toNaPP-buffer pH 7.2 over 10 column volumes. Qβ VLP for further processingeluted between 3.0 and 7.0 column volumes after the start of thegradient. Qβ VLP containing peak fractions were pooled and stored at 4°C.

Example 15

Chromatography on Immobilized Metal Ion Affinity Chromatography Matrices

A Chelating SEPHAROSE® Fast Flow column was loaded with Zn²⁺-ionsessentially according to the manufacturer's instructions in thefollowing way:

-   -   wash with deionized water: 5 CV    -   charge with 0.2 M ZnSO4: 5 CV    -   wash with deionized water: 5 CV    -   wash with elution buffer 1 (20 mM NaPP, 1 M NaCl, pH 5.0): 5 CV    -   wash with deionized water: 5 CV    -   sanitization: 0.5 M NaOH; contact time: 5 h    -   equilibration: 20 mM NaPP-buffer, 1 M NaCl, pH 7.2 (5 CV)

Chromatography on Chelating SEPHAROSE® Fast Flow was performed asfollows: 10 mg of a peak fraction from the separation on FRACTOGEL® EMDTMAE were loaded per ml bed volume of a column prepared according to theprocedure above and the flow through was collected. The loaded proteincould be recovered quantitatively in the flow through while residualproteins from the expression host were reduced significantly.

The invention claimed is:
 1. A process for the purification of a VLPfrom a recombinant bacterial host expressing said VLP, wherein said VLPis a VLP of RNA bacteriophage Qβ comprising coat protein of SEQ ID NO:3,the process comprising the steps of: (a) homogenizing said bacterialhost; (b) clarifying the homogenate obtained by said homogenizing; (c)purifying said VLP from the clarified homogenate obtained by saidclarifying in a first chromatography comprising the steps of: (i)binding said VLP to a first chromatography matrix; (ii) washing saidfirst chromatography matrix; and (iii) eluting said VLP from said firstchromatography matrix; and (d) further purifying said VLP from theeluate obtained by said first chromatography in a second chromatography,wherein said second chromatography is performed on a secondchromatography matrix, wherein said second chromatography matrix is ahydroxyapatite matrix; wherein said steps are performed in the givenorder.
 2. The process of claim 1, wherein said second chromatographycomprises the steps of: (i) binding said VLP to said secondchromatography matrix, wherein said second chromatography matrix is ahydroxyapatite matrix; (ii) washing said second chromatography matrix;and (iii) eluting said VLP from said second chromatography matrix;wherein said steps are performed in the given order.
 3. The process ofclaim 1, said process additionally comprising the step of finallypurifying said VLP obtained by said second chromatography by at leastone third chromatography, wherein said at least one third chromatographyis selected from: (a) hydrophobic interaction chromatography (HIC); (b)immobilized metal ions affinity chromatography (IMAC); and (c) sizeexclusion chromatography.
 4. The process of claim 1, wherein saidclarifying of said homogenate is performed by a method selected from thegroup consisting of: (a) centrifugation; (b) tangential flow filtration;and (c) a combination of (a) and (b).
 5. The process of claim 1, whereinsaid first chromatography matrix is an anion exchange matrix.
 6. Theprocess of claim 1, wherein said first chromatography matrix is atentacle anion exchange matrix comprising (i) resin particles ofcross-linked methacrylate polymer or cross-linked vinyl polymer (ii)acrylamide tentacles, wherein said acrylamide tentacles are attached tothe surface of said resin particles, and wherein said acrylamidetentacles are substituted with TMAE (Trimethylaminoethyl-) groups. 7.The process of claim 1, wherein said first chromatography comprises thesteps of: (i) equilibrating said first chromatography matrix with afirst equilibration buffer; (ii) binding said VLP to a firstchromatography matrix; (iii) washing said first chromatography matrixwith a first washing buffer; and (iv) eluting said VLP from said firstchromatography matrix with a first elution puffer; wherein said firstequilibration buffer, said first washing buffer and said first elutionbuffer comprise an alkaline metal halogenide.
 8. The process of claim 7,wherein said first equilibration buffer comprises at most about 200 mMsodium chloride, said first washing buffer comprises about 425 mM sodiumchloride, and said first elution buffer comprises least about 500 mMsodium chloride or a gradient of sodium chloride, wherein said gradientis from at most about 400 to at least about 650 mM sodium chloride. 9.The process of claim 7, wherein said first equilibration buffer, saidfirst washing buffer and said first elution buffer comprise a pH ofabout 7.2.
 10. The process of claim 1, wherein said hydroxyapatitematrix is a ceramic hydroxyapatite matrix.
 11. The process of claim 2,wherein said second chromatography comprises the steps of: (i)equilibrating said second chromatography matrix with a secondequilibration buffer; (ii) binding said VLP to said secondchromatography matrix, wherein said second chromatography matrix is ahydroxyapatite matrix; (iii) washing said second chromatography matrixwith a second washing buffer; and (iv) eluting said VLP from said secondchromatography matrix; wherein said second equilibration buffer, saidsecond washing buffer and said second elution buffer, comprise analkaline metal halogenide.
 12. The process of claim 11, wherein saidsecond equilibration buffer comprises about 100 to 400 mM sodiumchloride, said second washing buffer comprises about 150 mM sodiumchloride, and said second elution buffer comprises 900 mM sodiumchloride and about 200 mM sodium phosphate buffer.
 13. The process ofclaim 11, wherein said second equilibration buffer, said second washingbuffer and said second elution buffer comprise a pH of about 7.2. 14.The process of claim 1, wherein said clarifying further comprises thestep of exposing said VLP to oxidative conditions.
 15. The process ofclaim 1, wherein said at least one third chromatography is at least onesize exclusion chromatography.
 16. A process for the purification of aVLP of RNA bacteriophage Qβ from a recombinant bacterial host expressingsaid VLP, wherein said VLP comprises coat protein of SEQ ID NO:3, theprocess comprising the steps of: (a) homogenizing said bacterial host;(b) clarifying the homogenate obtained by said homogenizing, whereinsaid clarifying further comprises the step of exposing said VLP tooxidative conditions; (c) purifying said VLP from the clarifiedhomogenate obtained by said clarifying in a first chromatographycomprising the steps of: (i) equilibrating a tentacle anion exchangematrix, wherein said equilibrating is performed with a firstequilibration buffer, wherein said first equilibration buffer comprisesabout 150 mM sodium chloride and a pH of 7.2; (ii) binding said VLP tosaid tentacle anion exchange matrix; (iii) washing said tentacle anionexchange matrix, wherein said washing is performed with a first washingbuffer comprising about 425 mM sodium chloride and a pH of 7.2; and (iv)eluting said VLP from said tentacle anion exchange matrix, wherein saideluting is performed with a first elution buffer comprising a gradientof 425 to 650 mM sodium chloride and a pH of 7.2; (d) further purifyingsaid VLP from the eluate obtained by said first chromatography in asecond chromatography comprising the steps of: (i) equilibrating ahydroxyapatite matrix wherein said equilibrating is performed with asecond equilibration buffer comprising about 150 mM sodium chloride anda pH of 7.2; (ii) binding said VLP to hydroxyapatite matrix; (iii)washing said hydroxyapatite matrix, wherein said washing is performedwith a second washing buffer comprising about 150 mM sodium chloride anda pH of 7.2; (iv) eluting said VLP from said hydroxyapatite matrix,wherein said eluting is performed with a second elution buffercomprising about 900 mM sodium chloride, about 200 mM sodium phosphatebuffer and a pH of 7.2; (e) finally purifying said VLP contained in theeluate of said second chromatography by exactly one size exclusionchromatography, wherein said size exclusion chromatography is performedin the presence of about 150 mM sodium chloride, and wherein furthersaid size exclusion chromatography is performed using a Sepharose CL-4Bgel filtration matrix; wherein said steps are performed in the givenorder.
 17. The process of claim 1, wherein said first chromatographymatrix is a tentacle anion exchange matrix.
 18. The process of claim 1,wherein said first chromatography matrix is an anion exchange matrixcomprising TMAE groups.
 19. The process of claim 7, wherein saidalkaline metal halogenide is potassium chloride or sodium chloride.